Molecular mechanism of obesity-related left ventricular systolic dysfunction in mice and application of ultrasonic strain detection technology

Introduction

The incidence rate of obesity is increasing (1,2). Obesity and the various metabolic diseases caused by obesity have become serious threats to human health (3). However, accumulated clinical and anatomical data have shown that many patients have obesity before the occurrence of coronary atherosclerosis (4). Some of these patients show changes in cardiac structure and function, and clinical manifestations of heart failure begin to appear (5).

Conventional echocardiography cannot sensitively detect subclinical cardiac dysfunction in obese patients. The myocardial strain technique tracks myocardial acoustic spots frame by frame through speckle-tracking technology and obtains their motion trajectory to evaluate myocardial motion function. The advantages of this technique include that it has no angle dependence and can evaluate myocardial motion objectively and accurately (6). Additionally, the strain technique is able to detect subclinical left ventricular dysfunction at the early stages of many conditions.

Obesity-related abnormal metabolites activate the first line of defense of the body, and immune cells can accurately identify these abnormal metabolites. When the defense system is constantly stimulated by abnormal metabolites, it causes the circulatory system to secrete and release a substance called an exosome (7). An exosome mediates communication between organs and cells. Small cell vesicles transfer microRNA (miRNA) and regulate target messenger RNA in distant tissues (8-10). Obesity-related abnormal metabolites cannot activate human immune cells to secrete exosomes to transmit information (11). These abnormal metabolites activate nucleotide oligomerization domain-like receptor protein 3 (NLRP3) inflammatory bodies and upregulate the expression of inflammatory factors, which lead to myocardial remodeling and early myocardial injury. Through ultrasound strain technology, we can detect early myocardial contraction dysfunction such as reduced left ventricular longitudinal strain in obese mice even with normal left ventricular ejection fraction (LVEF). Metabolic disorders lead to myocardial microstructural remodeling and functional decline by activating inflammatory responses, promoting fibrosis, and other pathways. Our research focuses more on the mechanisms of myocardial dysfunction, and in the process of delving into the injury mechanisms of cardiac dysfunction, we have discovered clinically significant influencing factors.

This study aimed to detect changes in myocardial function in obese and healthy mice with a normal LVEF using the myocardial strain technique. The myocardial mechanism of the obese mice was analyzed by pathology, immunofluorescence detection, and exosome miRNA sequencing. We present this article in accordance with the ARRIVE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-340/rc).

Methods

Animal and study protocols

In this study, 30 male C57BL/6 mice (6-week-old, weighing 20–30 g) [Experimental Animal Center of Ningxia Medical University, Experimental Animal Production License Number: SCXK (Ning) 2020-0001] were selected and fed a high-fat diet (16% lipid, 0.25% cholesterol). The control group’s feed composition is 5% lipid, without adding cholesterol; all mice were free to drink water and feed. The animals completed a 12-week rearing period in a specific pathogen-free (SPF) barrier facility. barrier. In total, 15 obese mice with a body weight and Lee’s index greater than the mean ±1.5 times the standard deviation of the control group were selected as the obese group (12). Another 15 male C57BL/6 mice of the same age and sex as the experimental obese group were included as the control group. The procedures performed on the mice were approved by the Ethics Committee of General Hospital of Ningxia Medical University (No. IACUC-NYLAC-2020-067), in compliance with national guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Ultrasound biomicroscopy

The mice were anesthetized with 2% isoflurane and fixed in the supine position on a thermostatic platform with their limbs fixed. Electrodes were then attached for the electrocardiographic recording. The chest hair of the mice was removed before imaging using a chemical hair removal agent, and the echocardiographic data were collected after the onset of anesthesia and when the heart rate was stable. Ultrasound biomicroscopy was performed to dynamically observe the left ventricular ultrasound biology of the mice, including the morphology, and blood flow changes in mechanics, function, and strain. The morphological measurements included the thickness of the left ventricular septum, left ventricular internal diameter, and posterior left ventricular wall, the left ventricular end-systolic internal diameter, and the left ventricular end-diastolic internal diameter. These variables were measured by M-mode ultrasound. The LVEF was calculated using the Teichholtz correction formula. Radial and circumferential strains and strain rates were measured in the short-axis sections of the papillary muscle. Echocardiography strain software was used to automatically trace the endocardium to produce an overall myocardial strain and strain rate curve, which allowed the myocardial strain and strain rate parameters to be derived for each section.

Circulating exosome extraction and identification

Blood was taken from the mice after euthanasia, and the serum was collected by centrifugation. The serum was moved to a new centrifuge tube and centrifuged at 2,000 ×g for 30 min at 4 ℃. The supernatant was carefully transferred to another centrifuge tube and centrifuged at 10,000 ×g for 45 min at 4 ℃ and to remove larger vesicles. The supernatant was removed and filtered through a 0.45-µm membrane, and the filtrate was collected. The supernatant was removed, resuspended with 10 mL of pre-cooled 1× phosphate-buffered saline (PBS), and centrifuged again at 100,000 ×g for 70 min at 4 ℃ using an ultra-rotor. The supernatant was then removed and resuspended with 150 µL of pre-cooled 1× PBS.

We used 20 µL of the resuspended supernatant for the electron microscopy. Of which, 10 µL was used to determine the particle size, and 10 µL was used to observe the exosomes. We pipetted 10 µL of the sample onto a copper grid for 1 min and allowed it to precipitate, and filter paper was used to remove float. A volume of 10 µL of UO₂ acetate was added dropwise to a copper grid and allowed to precipitate for 1 min. The samples were dried at room temperature for several minutes. We used 100 kv for the electron microscopic imaging. The particle size and concentration of the exosomes were obtained when the samples were ready.

The samples were incubated at 37 ℃ for 30 min, and the absorbance was recorded at an optical density of 562 nm on an enzyme marker. The protein concentrations of the samples to be tested were calculated from a standard curve. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a concentration of 10% or 15% according to the protein size of the sample to be tested was used to separate the proteins. The gel was run at 80 V until the sample ran out of the gel concentrate, and then at 100 V until the bromophenol blue tracking dye reached the bottom of the gel.

After electrophoresis, the gel was removed, activated in a suitable amount of methanol for 20 s, and transferred to a “sandwich” in the order of sponge, filter paper, electrophoresis gel, membrane, filter paper, and sponge. The power was switched on, and the membrane was transferred at 300 mA. The transfer time was determined according to the size of the protein to be detected.

After sealing, the membranes were cut as required, immersed in the prepared primary antibody solution (antibody dilution ratio: 1:1,000) and incubated overnight at 4 ℃. The primary antibody was recovered, and the membrane was washed with 20 mL of Tris Buffered Saline with Tween (TBST) for 10 min at room temperature, and this was repeated 3 times.

The secondary antibody was selected according to the primary antibody. The secondary antibody (antibody dilution ratio: 1:5,000) was mixed in 5% skimmed milk TBST solution, and the membrane was soaked in the secondary antibody solution and incubated for 1 h at room temperature. The membrane was then washed with 20 mL of TBST for 10 min at room temperature, and this procedure was repeated 3 times. The polyvinylidene difluoride membrane was removed, drained, and laid flat on cling film with the protein side up. An equal volume of enhanced chemiluminescence A/B solution was added dropwise to the membrane for 5 min in the dark. The film was then placed in an imager, and the band intensity was quantified.

miRNA sequencing

For the miRNA sequencing of the exosomes, LC Sciences performed the preparation of the tagged miRNA-sequencing libraries, sequencing, and next-generation sequencing data analysis. The library was sequenced with the Illumina Hiseq 2500 SE50 platform. The raw reads were subjected to an in-house program, ACGT101-miR (LC Sciences), to remove the adapter dimers, junk, low complexity, and common RNA families (e.g., ribosomal RNA, transfer RNA, small-nuclear RNA, and small nucleolar RNA) and repeats. Subsequently, the unique sequences with lengths of 18–26 nucleotides were mapped to specific species precursors in miRBase 22.0 via a BLAST search to identify the known miRNAs and novel 3p- and 5p-derived miRNAs. A higher number of reads than the average copy number of the data set was used as the criterion to filter the high-level miRNAs. The R package multiMiR (version 3.12) was used for the miRNA target scanning and prediction, while the clustering analysis of the target genes was performed using the R package clusterProfiler. Total RNA was isolated with TRIzol reagent (9109; Takara, Japan). The library was sequenced with the Illumina NovaSeq 6000 PE150. The following criteria were applied to filter the differentially expressed genes: |logfold change| >1 and P value <0.05. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed with the R package clusterProfiler. The results were visualized by GOplot.

Hematoxylin and eosin (H&E), wheat germ agglutinin staining, and immunofluorescence staining of the frozen sections of heart muscle

The frozen sections were re-warmed for 10 min, fixed in 4% paraformaldehyde for 30 min, washed in water for 5 min, and stained with lignin as follows: The frozen sections were washed in distilled water for 5–10 min, 1 min 0.5% hydrochloric acid ethanol fractionation: 10 s/10 to 20 quick dips, distilled water wash: 2 min, 0.2% ammonia blue return: approximately. 40 s, distilled water wash: 2× 1 min, 0.5% eosin staining: 5 min, distilled water quick wash, dehydration, transparency, neutral gum seal. The frozen sections were placed in an oven at 58 ℃ for 2 h. For the fixation, the frozen sections were immersed in paraformaldehyde for 30 min; the sections were placed in an immunohistochemical wet box, and 20 µg/mL of proteinase K drop was applied to each specimen. The specimen was then washed 3 times with PBS. The specimen was covered with a drop of 20 µg/mL lectin solution in an immunohistochemistry wet box at 37 ℃ for 1 h. The lectin staining solution was washed away with PBS. Next, 4',6-diamidino-2-phenylindole staining was performed for 5 min, observed under a fluorescent microscope and photographed.

The frozen sections were fixed. After fixation, the sections were stored in PBS at 4 ℃ awaiting use. The sections were washed 3 times with PBS for 5 min each time. The cells were closed using a closure solution usually for 30 min, incubated at room temperature for 1 h or overnight at 4 ℃, and rinsed 3 times with PBST for 5 min each time. Indirect immunofluorescence required the use of secondary antibodies. The sections were incubated for 1 h at room temperature protected from light, rinsed 3 times with PBST for 5 min each time, and then rinsed 1 time with distilled water. A single drop of blocking agent was added, and the slides were sealed and examined by fluorescence microscopy.

Statistical methods

SPSS 26.0 software was used for the statistical analysis. The normally distributed measurement data are expressed are the x¯±σ. The comparisons between the two groups were conducted using the independent sample t-test. A P value <0.05 indicated a statistically significant difference.

Results

Ultrasound biomicroscopy

Ultrasound biomicroscopy showed that the left ventricular end-diastolic internal diameter did not differ significantly between the control and obese groups. LVEF were lower in the control group than the obese group (P<0.05, Tables 1,2 and Figure 1). The thickness of the left ventricular septum was significantly greater in the obese group than the control group (P<0.05). The 2-dimensional (2D) speckle-tracking technique revealed a significant difference in the longitudinal strain and circumferential strain between the control and obese groups (P<0.05). The longitudinal strain and circumferential strain parameters were lower in the obese group than the control group (all P<0.05, Tables 1,2 and Figure 1B-1E).

Figure 1 Gross anatomy of the heart in the two groups of mice. (A) Ultrasound biomicroscopic examination of the mouse myocardium. (B,C) LV global function derived from M-mode ultrasound biomicroscopy. (D,E) Standard LV segmentation in the mid-ventricular short-axis (left) view. LV, left ventricular.

H&E, wheat germ agglutinin, and immunofluorescence staining of frozen sections of myocardium

The H&E staining of the frozen sections of the myocardial tissue showed that the septum of the obese group was significantly thicker than that of the control group (P<0.05, Figure 2). The myocardial cells of the mice in the control group were neatly arranged, with normal interstitial spaces and no inflammatory exudation. Conversely, the myocardium of the mice in the obese group showed fibrosis with a small amount of lymphocyte infiltration (Figure 2). The myocardial cell area of the obese group was higher than that of the control group, and the cell gap of the obese group was enlarged and the cells were loosely arranged (Figure 3). Immunofluorescence staining showed that the expression of NLRP3 was significantly higher in the obese group than the control group (Figure 4).

Figure 2 Hematoxylin and eosin staining of a mouse heart. (A,B) The left ventricular septum of the mice in the obese group was thickened. Scale bar: 1 mm. (C,D) Myocardial fibrosis with mild lymphocyte infiltration was observed in the mice in the obese group. Scale bar: 200 µm.

Figure 3 WGA staining showed that the size of the cardiomyocytes of the mice in the obese group was greatly increased (scale bar: 50 µm). (C,E) WGA staining for obese group and control group, respectively; (B,F) DAPI staining for obese group and control group, respectively; (A) and (D) respectively represent the obese group and the control group. DAPI, 4',6-diamidino-2-phenylindole; WGA, wheat germ agglutinin.

Figure 4 Immunofluorescence staining revealed elevated NLRP3 expression in obese mice (scale bar: 200 µm). (A-D) Obese group; (E-H) control group. (A,E) NLRP3 staining (green); (B,F) WGA staining (red); (D,H) DAPI staining (blue); (C,G) Merge. DAPI, 4',6-diamidino-2-phenylindole; NLRP3, Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 3; WGA, wheat germ agglutinin.

Exosome isolation and identification

Most of the exosomes were between 90 and 230 nm in size. The exosomes were round or oval vesicles of an uneven size, and intact exosomes were observed under transmission electron microscopy (Figure 5). In addition, the exosome-specific markers CD9, CD63, and ALIX were significantly more abundant in these exosomes. Calnexin and dlyceraldehyde-3-phosphate dehydrogenase were not detected (Figure 5A). Taken together, these data indicated that the isolation of mouse circulating exosomes was successful.

Figure 5 Exosome identification and miRNA sequencing. (A,B) Successful extraction of exosomes. (C,D) There was a significant difference in the secreted miRNA expression between the obese group and the control group. (E,F) Data from TargetScan and microRNA.org databases for target gene prediction, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses of common target genes by bioinformatics software. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; miRNA, microRNA; NG, normal group; OG, obese group; TGF, transforming growth factor.

miRNA sequencing and bioinformatics analysis within the exosomes

After filtering and conducting a quality inspection of the original data, the two groups of differentially expressed miRNAs were counted, and the target genes co-expressed by these miRNAs were predicted and analyzed for function enrichment. The differentially expressed miRNAs between the two groups were selected at the following two levels: the difference multiple level: |log₂(fold change)| >1; and the significance level: false discovery rate ≤0.001. According to the results of the differential gene detection, a hierarchical cluster analysis was performed on the differential miRNAs. Hundreds of differentially expressed miRNAs were detected between the two groups. The expression of miRNA in the plasma exocrine of the obese and control groups was detected, and 455 differentially expressed miRNAs were found (of which 331 were upregulated and 124 were downregulated).

To further explore the functions of the differential miRNAs between the obese mice and control mice, the GO and KEGG databases were used to analyze the function and pathway enrichment of these target genes. The screening standard for effective enrichment was a Q value ≤0.05. The GO enrichment analysis of the target genes divided the terms into the following three categories: cellular components (CCs), molecular functions (MFs), and biological processes (BPs) (Figure 5). In terms of the enriched BPs, these genes were mainly involved in the processes of cell metabolism, proliferation, and death. In terms of the enriched MFs, these genes were mainly involved in the regulation of transcriptional activity and signal transduction activity.

A KEGG analysis was then conducted to annotate and classify the signaling pathways in which the target genes were involved (Figure 5). In terms of the enriched BPs, the target genes were mainly involved in signal transduction, cell growth and death, transport, and metabolism. In terms of the enriched CCs, the genes were mainly involved in the cell membrane and cytoplasm. In terms of the enriched MFs, the main functions included protein binding, metal ion binding, and nucleotide binding. In addition, the differentially expressed miRNA target genes were enriched in the cardiovascular system, immune system, nervous system, and other systems.

Discussion

Patients with obesity show altered cardiac structure and function before the development of coronary atherosclerosis (13,14) consistent with our research findings. Obesity-induced myocardial remodeling refers to changes in the myocardial structure, function, and phenotype, including changes in the ventricular weight, volume, or morphology, an increase in fibrosis content, cell hypertrophy, and cell death. The most sensitive change during cardiac remodeling is ventricular septal thickening.

In this study, ultrasonic biomicroscopy was used to observe the cardiac structure and it showed that the thickness of the left ventricular septum of the mice in the obese group was significantly greater than that of the mice in the control group (Figure 1B). Most of the clinical methods for measuring the LVEF are 2D. The measurement of the LVEF by 2D echocardiography has limitations related to mathematical geometry and morphology. However, using the myocardial strain technique, we found that the longitudinal strain and circumferential strain values of the mice in the obese group were lower than those of the mice in the control group. This finding suggests that early changes in cardiac morphology and a decrease in myocardial contractility in obesity lead to myocardial damage, and cardiac structure is altered in the early stage of obesity. In this study, wheat germ agglutinin staining showed that the cardiomyocyte area of the obese group was larger than that of the control group, which reflected the hypertrophy of the cardiomyocytes.

When obesity is present, the excessive deposition of lipids in white adipose tissue damages the body’s function and reduces the secretion of adipokines (15,16). Additionally, pro-inflammatory factors, such as tumor necrosis factor-α and interleukin-(IL)1, IL-1β, and 1L-6, are released from the adipose tissue of the epicardium and other organs of the circulatory system, which may lead to systemic inflammation (17). The NLRP3 inflammasome induces the release of inflammatory mediators and participates in the pathogenesis of the inflammatory response of atherosclerosis during development (18). The NLRP3 inflammasome has been shown to activate IL-1β and IL-18 (19), promote the occurrence and development of a variety of metabolic diseases, block their biological effects, and effectively alleviate the progress of metabolic diseases (20). In this study, the H&E staining of frozen sections of myocardial tissue showed a small amount of lymphocyte infiltration in the obese group. Additionally, NLRP3 expression in the obese group was higher than that in the control group. These findings indicate that myocardial injury occurs in the early stage of obesity, an inflammatory response occurs, and the heart is remodeled.

The related abnormal metabolites caused by obesity first activate the body’s first defense system (i.e., innate immunity). The innate immune cells can accurately identify any abnormal metabolites, and then activate a series of self-defense systems (21). Because the defense system is continuously stimulated by abnormal metabolites, these stimulation signals exchange information between cells, which causes the cardiomyocytes to release exosomes. However, the difference in the exosome composition between obese mice and normal mice is still unknown. Thus, miRNA expression in the plasma exocrine of the obese and normal groups was detected, and 455 differentially expressed miRNAs were found (of which 331 were upregulated and 124 were downregulated).

To further explore the function of the differential miRNAs between the obese mice and normal mice, the GO and KEGG databases were used to analyze the function and pathway enrichment of these target genes. It was found that these genes were mainly involved in the processes of cell metabolism, proliferation, and death. Their MFs were enriched in the regulation of transcriptional activity and signal transduction activity. At the same time, the signaling pathways involved in these target genes were annotated and classified by the KEGG pathways (Figures 4,5B). The nuclear factor kappa B (NF-κB) signaling pathway accounts for the main part.

A study has shown that in obese patients, the activation of NLRP3 inflammatory bodies is strictly regulated by NF-KB (22). In this study, the expression of the NLRP3 inflammatory bodies was increased in the obese mice, which suggests that NLRP3 inflammatory bodies may be related to left ventricular remodeling in obese mice. NLRP3 was significantly enhanced in the left ventricular myocardium of the obese mice compared to the control mice. NLRP3 inflammasome is associated with the increased lipotoxicity of intracellular ceramide, which induces caspase-1 cleavage in macrophages and adipose tissue (23). In obese rodent models, caspase-1 and fibrosis were found to be significantly enhanced, which was associated with a significant decrease in LVEF (24). This is one of the reasons for left ventricular systolic dysfunction in obese mice.

In this study, we further demonstrated that there are a series of miRNAs in the obese circulatory system, which help to mediate the signaling pathways related to the inflammatory response, and our data suggest that circulating EVs may mediate the myocardial inflammatory response by regulating NF-κB. Thus, it is of great significance to clarify the role and mechanism of the cardiomyocyte NLRP3 regulatory pathway in myocardial injury in the early stage of obesity, and it may serve as a target for the prevention and treatment of metabolic diseases caused by obesity.

Conclusions

The myocardial strain technique detected changes in myocardial systolic function earlier than the LVEF in the obese mice. Left ventricular remodeling and an NLRP3-mediated inflammatory response occurred in the obese mice. The expression of miRNA in the plasma exocrine of the obese and control groups was detected, and 455 differentially expressed miRNAs were found (of which 331 were upregulated and 124 were downregulated). Further functional verification of the major miRNAs might provide new ideas and targets for the risk assessment, prevention, and treatment of obesity-related cardiovascular diseases.

Acknowledgments

This abstract was presented at the Cardiovascular Innovations and Applications The 33rd Great Wall International Congress of Cardiology Asian Heart Society Congress 2022 Meeting in Beijing China. We would like to thank Ellen Knapp, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn/), for editing the English text of a draft of this manuscript.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-340/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-340/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-340/prf

Funding: This work was financially supported by the Ningxia Natural Science Foundation (Nos. 2022AAC03481 and 2023AAC02060).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-340/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The procedures performed on the mice were approved by the Ethics Committee of General Hospital of Ningxia Medical University (No. IACUC-NYLAC-2020-067), in compliance with national guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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