Improvements in left ventricular regional and global systolic function following treatment with S100A4-shRNA after myocardial infarction in mice

Introduction

Myocardial infarction (MI) is a leading cause of death worldwide (1). Although statins, anticoagulants (2), and percutaneous coronary intervention are widely used (3), heart failure still occurs after various kinds of adverse cardiac remodeling post-MI (4). Cardiac fibrosis plays an important role in cardiac remodeling, leading to left ventricular (LV) systolic and diastolic dysfunction (5). However, effective diagnose and therapy for cardiac fibrosis remain undeveloped.

Echocardiography is a diagnostic method that can meet both clinical and basic medical needs because of its non-invasive, cost-effective, and time-saving nature (6). However, its occasional imprecise evaluation of disease severity and prognosis has necessitated its reformation (7). Recently, a novel technique named two-dimensional speckle tracking echocardiography (STE) has matured (8) with the ability to dramatically improve the accuracy in assessing cardiac performance based on myocardial strain analysis (9). Strain analysis provides integrated and detailed information with much higher sensitivity and specificity by capturing segmental tissue motions on multiple planes and axes (10).

S100 calcium-binding A4 (S100A4), also known as fibroblast-specific protein 1 (Fsp1), is unregulated in fibrotic diseases of the lung (11), liver (12), kidney (13) and heart (14). Coming from the S100 gene family (15), it is involved in immune response, differentiation, cytoskeleton dynamics, and cell growth (16). Our previous study has found that downregulation of S100A4 alleviates cardiac fibrosis via Wnt/β-catenin pathway in mice, which may provide a potential therapeutic target for cardiac fibrosis after MI (17). However, more animal experiments, safety assessments, and efficient myocardial transfections are needed to enhance our findings.

In this study, we investigated the role of two-dimensional STE in the early assessment of myocardial function after MI, detected the potential parameters of stain analysis, and verified the efficacy and safety of S100A4-shRNAin cardiac fibrosis in murine MI model.

Methods

Animal and ethics

All experiments were approved by the Live Animals Committee in Teaching and Research at Nanjing Medical University (Approval ID: IACUC-1703039). The study was performed in accordance with the guidelines and principles for the care and use of laboratory animals published by the National Institutes of Health (No. 85-23, revised 1996).

C57BL/6 mice (6 weeks old) weighing 20–25 g was obtained from Nanjing University Model Animal Research Center. They were kept in a 12 h/12 h light/dark cycle at a room temperature for least 10 days and fed with a standard diet before the experiment. Baseline vital signs and myocardial function were recorded.

Study protocol

A total of 48 male mice were randomly assigned to sham+S100A4-shRNA, sham+scrambled (Scr) sequence-shRNA, MI+S100A4-shRNA, and MI+Scr-shRNA groups (n=12 per group) by controlling two independent variables (MI and S100A4-shRNA administration). To detect LV regional and global systolic changes, we assessed cardiac function via both M-mode tracing and strain analysis at baseline, day 7, 14, and 28 after injection. Then, mice were sacrificed for further histolytic study at the 28^th day post echocardiography.

Surgery and gene transfection

Mice were anesthetized with 0.5% sodium pentobarbital (100 mg/kg) and then intubated and ventilated at 120 bpm during the operation. In the MI groups, the left anterior descending (LAD) coronary artery was ligated at 2 mm from the tip of the left auricle with 7–0 silk suture. After that, MI was confirmed by S-T segment elevation on an electrocardiogram. A volume of 5×10⁵ pfu/g Scr-shRNA or S100A4-shRNA was intramyocardially injected into 5 parts bordering the infarction zone or normal region via a 30-gauge Hamilton needle. The sham groups underwent chest open operation but no LAD ligation, and received the same dose and positions of Scr-shRNA or S100A4-shRNA administration shortly after the surgical procedure. The optimal transfection concentration (2.5 µL/g, 2×10⁷ pfu/mL) was determined by GFP expression rate and cell viability.

M-mode echocardiography

A Vivid 7 ultrasound (GE, Horten, Norway) equipped with an il3L intraoperative linear probe at 10.0–14.0 MHz was used in the study. The mice were imaged under light sedation and decreasing ambient lighting at 22–24 °C by an experienced handler. M-mode parameters were obtained at the mid-papillary level in the short-axis parasternal view, including inter ventricular septal diameter (IVSd), left ventricular posterior wall thickness (LVPWd), left ventricular internal diameter at diastole (LVIDd), left ventricular internal diameter at systole (LVIDs), ejection fraction (EF), and fractional shortening (FS).

Strain analysis

Circumferential and radial strain were analyzed from 3 consecutive cardiac cycles of the mid-LV (parasternal papillary muscle level) short axis images by Echopac PC software (version 113.1, GE, Horten, Norway). Region of interest was measured by manually contouring the area between endocardial and epicardia borders. Grayscale images were then analyzed following frame-to-frame movement of stable patterns of natural acoustic markers (or speckles) over each cardiac cycle with frame rates ranging from 200 to 300 fps. Finally, the values of peak radial strain (pRS) and time to peak radial strain (pRSt) were derived from 6 segments by strain analysis.

Masson trichrome staining

The LV tissues were fixed in 4% buffered formalin, embedded in paraffin, and then prepared into 5-µm-thick sections. Masson staining was performed to investigate the distribution and assess the extent of myocardial interstitial fibrosis.

Immunohistochemistry (IHC)

The tissues were prepared into 5-µm-thick paraffin sections with deparaffinization and antigen retrieval in 1 mM EDTA (pH 9.0) for 15 min. Then, slides were applied with 5% bovine serum albumin (BSA) at room temperature for 1 h, and incubated with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 30 min. Before mounting, chromogens (diaminobenzidine or 3-amino-9-ethylcarbazole) and hematoxylin were used for counterstain.

Statistical analysis

All experiments were repeated and obtained with repetitions of qualitatively similar data. All data were analyzed by SPSS 17.0 (Chicago, IL, USA) and GraphPad Prism 6.0 software (CA, USA). Intergroup comparison of the continuous numerical variables was tested by t-test or two-way ANOVA. Values were expressed as mean ± standard deviation, and P<0.05 was considered statistically significant.

Results

Basic characteristics

A total of 48 male mice were randomly assigned to study groups and treatment regimes. There was no significant difference in the vital baseline signs and cardiac function between the sham+Scr-shRNA, sham+S100A4-shRNA, MI+Scr-shRNA, and MI+S100A4-shRNA groups (all P>0.05) (Table 1).

Table 1 Baseline characteristics of study animals
Full table

M-mode echocardiography detected LV deformation after MI

We discovered significant change in the LV deformation before and at day 7 and 28 post-MI (all n=24) (Figure 1A). M-mode tracings showed the values of EF (77.83%±3.24% vs. 37.33%±3.50%, P<0.01) and FS (40.33%±2.43% vs. 18.50%±0.96%, P<0.01) statistically decreased at the 7^th day compared with the baseline group. After that, EF (44.17%±2.67% vs. 37.33%±3.50%, P<0.01) increased on the 28^th day, compared with the 14^th day tracings (Figure 1B). In addition, compared with day 14, FS (22.33%±2.43% vs. 18.50%±0.96%, P<0.01) significantly increased on day 28 (Figure 1C). M-mode imaging showed cardiac function dropped after MI and improved after 1-month repatriation.

Figure 1 Cardiac function diminution via standard echocardiography. (A) M-mode echocardiography indicated cardiac deformation between the day 14 and day 28 groups. (B) EF values decreased between the 7^thvs. 0 day group and increased until the 28^th day. (C) Compared with the day 14 group, FS values significantly increased in the day 28 group. EF, ejection fraction; FS, fractional shortening; P values were obtained using Student’s t-test; **, day 7 vs. baseline group, P<0.01; day 28 vs. day 14 group, P<0.01; n=24 per group.

Early identification by STE of cardiac changes in pRS and pRSt post-MI

To detect the changes of LV regional systolic function after MI, radial strain analysis was performed via two-dimensional STE (all n=24, Table 2). Examples showed a significant increase of LV systolic function in the 14^th day compared with the day 7 group (Figure 2A). A marked increase of pRS (anteroseptal, 23.83%±1.12% vs. 20.25%±1.02%, P<0.01; anterior, 23.17%±1.03% vs. 20.08%±1.08%, P<0.05; inferoseptal, 22.92%±1.15% vs. 19.58%±1.03%, P<0.05) was detected between the 7^th and 14^th day, especially in the LV anteroseptal wall (Figure 2B). Additionally, pRSt statistically decreased (anteroseptal 76.75±3.18 vs. 92.00±3.69 ms, P<0.05; anterior, 76.92±2.64 vs. 91.42±2.52 ms, P<0.05; inferoseptal, 74.58±2.60 vs. 88.42±2.09 ms, P<0.05) in the day 14 group compared with the day 7 group (Figure 2C). The results revealed that STE identified positive changes of LV systolic function on day 14 post-MI, which is earlier than that in the M-mode tracings.

Table 2 Left ventricular systolic function detected after MI by speckle tracking echocardiography
Full table

Figure 2 Earlier identification by STE of cardiac remolding after MI. (A) Examples of left ventricular myocardial function assessments as shown by strain analysis at day 7 and day 14; (B) marked increase of peak radial strain indicated by STE between the 14^th and 7^th day groups; (C) time to peak radial strain decreased in anteroseptal wall in the day 14 group, compared with the day 7 group. P values were obtained using Student’s t-test; *, day 28 vs. day 14 group, P<0.05; **, day 7 vs. baseline group, P<0.01; day 28 vs. day 14 group, P<0.01; n=24 per group. STE, speckle tracking echocardiography; MI, myocardial infarction.

S100A4-shRNA improves LV systolic function after MI

To detect the efficacy of S100A4-shRNA in attenuating regional deformation among sham+Scr-shRNA, sham+S100A4-shRNA, MI+S100A4-shRNA, and MI+Scr-shRNA groups, M-mode echo and strain analysis were used at the 28^th day post-surgery (n=12 per group, Table 3). M-mode echo showed both EF (50.50%±0.72% vs. 43.42%±0.82%, P<0.05) (Figure 3A) and FS (27.42%±0.71% vs. 21.92%±0.77%, P<0.05) (Figure 3B) increased in the MI+S100A4-shRNA group, compared with the MI+Scr-shRNA group. STE showed significant elevation of pRS (29.00%±0.87% vs. 24.67%±0.71%, P<0.05) in the anteroseptal segment (Figure 3C), and a decline of pRSt (60.67±1.43 vs. 73.42±3.54 ms, P<0.05) in the LV anteroseptal wall (Figure 3D) in the MI+S100A4-shRNA group, compared with the MI+Scr-shRNA group. The results demonstrated that S100A4-shRNA was effective in alleviating LV systolic function after MI.

Table 3 Left ventricular global and regional systolic function detected at day 28 post-surgery
Full table

Figure 3 STE and M-mode echo detected the efficacy of S100A4-shRNA in cardiac fibrosis. (A) M-mode echo showed EF increased in the MI+S100A4-shRNA group, compared with the MI+Scr-shRNA group. (B) FS was elevated in the MI+S100A4-shRNA group, compared with the MI+Scr-shRNA group. (C) STE showed significant elevation of radial strain in the MI+S100A4-shRNA group. (D) Time to peak radial strain declined in the MI+S100A4-shRNA group compared with the MI+Scr-shRNA group. P values were obtained using Student’s t-test or two-way ANOVA. **, MI+S100A4-shRNA vs. MI+Scr-shRNA group, P<0.01; MI+Scr-shRNA group vs. sham+Scr-shRNA group, P<0.01; n=12 per group. STE, speckle tracking echocardiography; MI, myocardial infarction.

S100A4-shRNA alleviated cardiac fibrosis after MI

Western blotting showed the expression of α-SMA (Figure 4A,B), collagen I (Figure 4A,C) protein decrease (fibrosis markers), and S100A4 level decline (Figure 4A,D) in the MI+S100A4-shRNA group, compared with the MI+Scr-shRNA group. The extent of cardiac fibrosis reduced in the MI+S100A4-shRNA group (18.91%±2.38% vs. 32.39%±3.06%) according to representative Masson’s trichrome staining (Figure 4E). Immunohistochemistry showed that the levels of α-SMA decreased in the MI+S100A4-shRNA group (15.80%±1.49% vs. 27.13%±2.41%) (Figure 4F). The results revealed the role of S100A4-shRNA in attenuating cardiac fibrosis after MI.

Figure 4 S100A4-shRNA ameliorated cardiac fibrosis after MI. (A,B) Western blotting showed the expression of α-SMA decreased in the MI+S100A4-shRNA group compared with the MI+Scr-shRNA group. (A,C) The expression of collagen I reduced in the MI+S100A4-shRNA group as indicated by western blotting. (A,D) Western blotting showed the S100A4 level declined in the MI+S100A4-shRNA group. (E) Masson’s trichrome staining showed that the extent of cardiac fibrosis decreased in the MI+S100A4-shRNA group. (F) Immunohistochemistry showed the levels of α-SMA decreased in the MI+S100A4-shRNA group. P values were obtained using Student’s t-test or two-way ANOVA. **, MI+S100A4-shRNA vs. MI+Scr-shRNA group, P<0.01; MI+Scr-shRNA group vs. sham+Scr-shRNA group, P<0.01; sham+S100A4-shRNA group vs. sham+Scr-shRNA group, P<0.01; n=12 per group. MI, myocardial infarction.

Discussion

We investigated the role of two-dimensional STE in early and comprehensive assessment of regional LV function after MI, and assessed the efficacy and safety of S100A4-shRNA in cardiac fibrosis after MI in mice. Firstly, M-mode imaging verified LV systolic function significantly declined after MI, and then improved 1 month later (Figure 1). Secondly, STE was more effective in the early assessment of cardiac deformation on the 14^th day post-MI compared with conventional echocardiography (Table 2, Figure 2). Furthermore, LV global and regional systolic function statistically improved in the MI+S100A4-shRNA group by M-mode echo and strain analysis (Table 3, Figure 3). In addition, western blotting, Masson’s trichrome staining, and immunohistochemistry showed the efficacy of S100A4-shRNA in attenuating cardiac fibrosis after MI (Figure 4).

In the study, we qualified two new parameters for radial strain-based analysis: pRS and pRSt. They could reliably detect cardiac dysfunction in the LV anteroseptal, anterior, and inferoseptal walls after LAD coronary artery ligation. STE is a novel technology for analyzing motion and has abundant indicators (18), even though some of these indicators from strain analysis are considered useless in analyzing the small heart of target animals (19). Studies have proven circumferential strain to be highly sensitive in detecting cardiac function in mice (20); however, a comprehensive evaluating system for STE has not been conducted yet. Thus, our findings will be beneficial in constructing a new STE assessment system.

Previous research commonly used clinical echocardiography (with frequencies of up to 15 MHz) and simple parameters, like LV size (21). Therefore, the statistical data on circumferential and longitudinal strain in our study were not analyzed because of the low ultrasound frequency and image quality. However, studies with high frequency transducers (30 MHz) have shown their reliability and reproducibility in assessing both global and regional function in the post-MI remodeling of mouse hearts (22-24). Further studies are needed to assess the feasibility and accuracy of high-frequency two-dimensional echocardiography.

Our previous experiments have found that S100A4-shRNA significantly inhibits cardiac fibroblasts differentiation after MI (25). This study provides more evidence of S100A4-shRNA in attenuating myocardial dysfunction and cardiac fibrosis after MI. Nevertheless, the pathophysiology underlying this attenuation and the association between LV remodeling and cardiomyocyte apoptosis remain to be determined. Moreover, with the development of ultrasound targeted microbubble destruction (26), more efforts should be taken to transfect target agents more efficiently and noninvasively.

Conclusions

S100A4-shRNA can be utilized as a therapeutic target to improve regional deformation and attenuate cardiac fibrosis following MI. Two-dimensional STE is useful in the early and comprehensive assessment of LV systolic function in mice.

Acknowledgments

Funding: This study was supported by the National Natural Science Foundation of China (Grant No. 81871359), by Jiangsu Provincial Key Discipline of Medicine (ZDXKA2016003), by the Natural Science Foundation of Jiangsu Province (BK20161057), by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), by the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX18_1478), by the Postgraduate International Exchanges and Cooperation Project of Nanjing Medical University (C090), and the China Scholarship Council (201808320318).

Footnote

Conflicts of Interest: The authors have no conflicts of interest to declare.

Ethical Statement: All experiments were approved by the Live Animals Committee in Teaching and Research at Nanjing Medical University (Approval ID: IACUC-1703039). The study was performed in accordance with the guidelines and principles for the care and use of laboratory animals published by the National Institutes of Health (No. 85-23, revised 1996).

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