Dapagliflozin Alleviates Coxsackievirus B3-induced Acute Viral Myocarditis by Regulating the Macrophage Polarization Through Stat3-related Pathways

Six-week-old male BALB/c mice were purchased from Vital River Laboratory Animal Technology (Beijing, China). All animals were fed in the Experimental Animal Center of Shandong Provincial Hospital (Shandong, People’s Republic of China). All animal experiments and procedures were approved by the Ethics Committee of Shandong Provincial Hospital, and the experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edition, published by the National Institutes of Health (NIH Publication No. 85–23, revised 1996) [22]. The CVB3 (Nancy strain) was prepared by passage through HeLa cell cultures. Virus titers were determined through a 50% tissue culture infectious dose (TCID50) assay of HeLa cells and calculated by the Reed–Muench method [23].

Forty-eight male mice (18–20 g, 6 weeks old) were randomly assigned into 4 groups with 12 mice per group: Control, Dapa (dapagliflozin, a specific SGLT2 inhibitor, Bristol-Myers Squibb), CVB3, and the combination of CVB3 + Dapa. Mice were injected with 100 µl of phosphate-buffered saline (PBS) containing 10³ TCID50 of CVB3 intraperitoneally at day 0 to establish a VMC model as previously described in the literature, while control mice were injected with 100 µl of PBS. Dapagliflozin (0.1 mg/kg per day) dissolved in 60% propylene glycol or control agent (60% propylene glycol) was given daily by gavage from day 1. To further investigate whether dapagliflozin alleviates myocarditis by increasing stat3 phosphorylation, 36 male mice (18–20 g, 6 weeks old) were injected 10³ TCID50 of CVB3 intraperitoneally at day 0 to establish a VMC model, and then randomized to either CVB3, CVB3 + Dapa, or CVB3 + Dapa + STATTIC. STATTIC (MedChemExpress,10 mg/kg) was given every 2 days intraperitoneally according to the instructions from day 1. At day 8, all the mice underwent echocardiography and were then executed, heart tissues and serum were taken for the next analysis.

The Raw264.7 macrophage cell line was cultured in RPMI-1640 (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Life Technologies). 1 × 10⁶ cells/well were seeded in 6-well plate and incubated in serum free medium overnight before treatment. To examine the effect of dapagliflozin, lipopolysaccharides (LPS) (Sigma) were administered to stimulate M1 macrophage activation, dapagliflozin (10 μM) was given at the same time; after 24 h stimulation, the macrophages’ phenotype distribution and inflammatory factors were observed.

Mouse cardiac echocardiography was operated in accordance with the manufacturers’ instructions. Mice were anesthetized by 2% isoflurane (supplied by Shandong Provincial Hospital). Then, the cardiac function parameters such as left ventricular internal dimensions in systole (LVID-s) and diastole (LVID-d), left ventricular fractional shortening (LVFS), and left ventricular ejection fraction (LVEF) were measured. The experiment was conducted in a double-blind fashion.

Serum cardiac troponin I (cTNI) activities were tested by Shandong Provincial Hospital. The levels of serum IL-1β, IL-6, and TNF-α were determined by ELISA (eBioscience) following the manufacturer’s instructions. All samples were measured in triplicate.

Heart samples were fixed in 10% buffered formalin solution and then embedded in paraffin. The samples were sectioned (5 μm thick) and then stained with hematoxylin and eosin (Servicebio, Wuhan,China) according to the manufacturing instructions, and the degree of inflammation was determined under 200 × magnification light microscopy. Myocardial histopathologic findings were quantified and scored for severity as follows: 0 = no inflammation, 1 = one to five distinct mononuclear inflammatory foci with 5% involvement or less of the cross-sectional area, 2 = more than five distinct mononuclear inflammatory foci or over 5% but not exceeding 20% involvement of the cross-sectional area, 3 = diffuse mononuclear inflammation involving over 20% of the area without necrosis, and 4 = diffuse inflammation with necrosis. The analysis was performed in a double-blinded manner by a trained pathologist. Besides, myocardial fibrosis was determined by Masson staining (Servicebio, Wuhan,China) according to the manufacturing instructions. Myocardial cells were stained red and collagenous fibers were stained blue. Collagen area fraction (collagen area/field area × 100%) was calculated by the Image-Pro Plus analysis system.

Heart samples were fixed in 10% buffered formalin solution and then embedded in paraffin. The samples were sectioned (5 μm thick). Incubate sections in 2 changes of xylene, 15 min each. Dehydrate in 2 changes of pure ethanol for 5 min, followed by dehydrating in gradient ethanol of 85% and 75% ethanol, respectively, 5 min each. Wash in distilled water. Immerse the slides in EDTA antigen retrieval buffer (pH 8.0) and maintain at a sub-boiling temperature for 8 min, standing for 8 min and then followed by another sub-boiling temperature for 7 min. Let air cool. Wash three times with PBS (pH 7.4) in a Rocker device, 5 min each. Block endogenous peroxidase: wash three times with PBS (pH 7.4) in a Rocker device, 5 min each eliminate obvious liquid, mark the objective tissue with liquid blocker pen. Immerse in 3% H₂O₂ and incubate at room temperature for 25 min, kept in a dark place. Then, wash again three times with PBS (pH 7.4) in a Rocker device, 5 min each. Eliminate obvious liquid, and mark the objective tissue with liquid blocker pen. Cover objective tissues with 10% donkey serum at room temperature for 30 min. Incubate slides with CD68 antibody (diluted at 1:200; Servicebio) overnight at 4 °C, wash slides three times with PBS (pH 7.4) in a Rocker device, 5 min each. Cover objective tissue with Cy3 conjugated Goat Anti-Rabbit IgG (diluted at 1:200; Servicebio), and incubate at room temperature for 50 min in dark conditions. Wash slides three times with PBS (pH 7.4) in a Rocker device, 5 min each. Incubate slides with TSA-FITC solution for 10 min in dark conditions. After that, wash slides three times with TBST in a Rocker device, 5 min each. Immerse the slides in EDTA antigen retrieval buffer (pH 8.0) and maintain at a sub-boiling temperature for 8 min, standing for 8 min and then followed by another sub-boiling temperature for 7 min, to remove the antibodies combined with tissue. Incubate slides with CD163 antibody (diluted at 1:1000; Servicebio) overnight at 4 °C, placed in a wet box containing a little water. Wash slides three times with PBS (pH 7.4) in a Rocker device, 5 min each. Then throw away the liquid slightly. Cover objective tissue with Cy3 conjugated Goat Anti-Rabbit IgG (diluted at 1:200; Servicebio), incubate at room temperature for 50 min in dark conditions. Eliminate obvious liquid, incubate slides with spontaneous fluorescence quenching reagent for 5 min, then wash slides under flowing water for 10 min. Incubate with DAPI solution at room temperature for 10 min, kept in a dark place. Wash three times with PBS (pH 7.4) in a Rocker device, 5 min each. Throw away liquid slightly, then coverslip with anti-fade mounting medium. Microscopy detection and collect images by Fluorescent Microscopy. DAPI glows blue by UV excitation wavelength 330–380 nm and emission wavelength 420 nm; FITC glows green by excitation wavelength 465–495 nm and emission wavelength 515–555 nm; CY3 glows red by excitation wavelength 510–560 nm and emission wavelength 590 nm.

Total RNA samples of myocardial infiltrating macrophages and cultured RAW264.7 were extracted with TRIzol reagent (Takara, China) in accordance with the manufacturer’s instruction. First-strand complementary DNA was synthesized using 1 μg of total RNA in a 20-µL reaction buffer containing MMLV-RT and oligo (dT) primers (Takara, China). The mixture was incubated at 42 °C for 60 min, 70 °C for 15 min, and then cooled to 4 °C. To detect the expression of genes (TNF-α, iNOS, CD206, Arginase-1 and Stat3), cDNA was amplified with specific real-time PCR primers by using SYBR green real-time PCR kits (Takara, China). The following mRNA primer sequences were used:

The quantified data were analyzed using the 2^−ΔΔCt method [24].

Fresh myocardial tissue was lysed by RIPA lysis buffer (Nanjing, China) and proteins were extracted. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membrane was closed with 5% bovine serum albumin and then incubated with the appropriate primary antibody overnight at 4 °C, followed by washing with TBS-Tween and incubation with the appropriate secondary antibody at room temperature for 1 h. Spots were detected using an ECL system (Amersham, UK) and optical density was measured using ImageJ software. The following antibodies were used: iNOS (Cell Signaling Technology, 13120S), CD206 (Abcam, ab125028), (Abcam, ab28946), IL-6 (Cell Signaling Technology, 12912S), Stat3 (Cell Signaling Technology, 9139S), Phospho-Stat3 (Cell Signaling Technology, 9145S), GAPDH polyclonal antibody (Proteintech, No.10494–1-AP).

As expected, the symptoms of viral myocarditis in the CVB3 group were evident, including a rough gray color, reduced activity and diet, and dyspnea at rest. These signs were improved to varying degrees after dapagliflozin treatment. We also monitored weight loss, the bodyweight of the CVB3 group decreased continuously from day 2 post-infection compared to the control group, and dapagliflozin has no effect on this change (Fig. 1a). No mice died in the control and Dapa groups, whereas 7 mice died in CVB3 group and 5 mice died in CVB3 + Dapa group. Dapagliflozin treatment significantly improved the survival rate of mice. (Fig. 1b). Taken together, dapagliflozin application significantly prevented the progression of CVB3-induced general presentation and improved survival.

We further assayed the potential of dapagliflozin to inhibit CVB3-mediated cardiac dysfunction by echocardiography on day 8. As Table 1 depicted, CVB3 infection led to significantly reduced cardiac contractility and impaired diastolic function, while dapagliflozin administration could reverse cardiac function decline. Thus, these data support the notion that dapagliflozin participates in CVB3-induced myocardial dysfunction in vivo.

Next, we examined the protective role of dapagliflozin against myocarditis-affected inflammation and cardiac remodeling in vivo on day 8. As shown in Fig. 1c, virus myocarditis elicited typical cardiac histopathological changes in the myocardium. Severe inflammatory lesions infiltrated by large numbers of inflammatory cells were observed, while dapagliflozin treatment significantly reduced the infiltration of inflammatory cells. Further quantitative evaluation revealed a significant reduction of mean pathological myocarditis scores in the CVB3 + Dapa group compared with the CVB3 group during the acute phase of virus myocarditis (P < 0.001, Fig. 1d). Consistent with these findings, the serum cTnI activities were also reduced in the mice treated with dapagliflozin (P < 0.001, Fig. 1g). For evaluation of cardiac remodeling, heart sections were stained with Masson trichrome dye to visualize interstitial collagen content. As shown in Fig. 1e, CVB3 resulted in a visible fibrosis in interstitial tissue of myocardium in the CVB3 group. Quantification of fibrosis (Fig. 1f) also showed a statistical increase in collagen deposition of virus mice. Contrary to this, in dapagliflozin treatment mice, the collagen area fraction was significantly lower than that in the CVB3 group (P < 0.001, Fig. 1e, f). Taken together, our data indicate that dapagliflozin protected mice against the pathological changes of viral myocarditis.

The phenotype of macrophages is directly regulated by the composition of the microenvironment, including cytokine profiles. We therefore examined the levels of inflammatory or anti-inflammatory cytokines in each group. Plasma samples were collected at day 8 and analyzed by ELISA. The levels of IL-1β, IL-6, and TNF-α, cytokines related to macrophage, were higher in the virus group than in the control group, but were lower in dapagliflozin-treated mice (Fig. 1h, i, j). To identify the subtype of infiltrated macrophages in myocardium, the marker for M1 (CD68+) and M2 (CD163+) was examined. Immunofluorescence showed that there were a large number of CD68 + macrophages infiltrated in the CVB3 groups, and CD163 + macrophages were more frequent in the CVB3 + Dapa group (Fig. 2a). Besides, the mRNA expression of iNOS (a marker of M1 macrophages) were significantly upregulated in the myocardial tissue of mice in the CVB3 group compared with the control group (Fig. 2b), which was significantly downregulated in the dapagliflozin-treated group compared with the CVB3 group (Fig. 2b). In addition, we found that the mRNA expression of CD206 (a marker of M2 macrophages) was significantly decreased in VMC mice, and dapagliflozin treatment alleviated this change (Fig. 2c). Besides, the level of iNOS and IL-6 were significantly upregulated in the myocardial tissue of mice in the CVB3 group compared with the control group, while it was significantly downregulated in the CVB3 + Dapa group compared with the CVB3 group (Fig. 4c, d). These results suggest that dapagliflozin decreased the percentages of M1 and increased the percentages of M2 in VMC mice.

Previous studies have demonstrated that Stat3 is important in regulating the phenotype of macrophages, here we investigate the effect of dapagliflozin on Stat3 in VMC. We found that pstat3/stat3 was significantly upregulated in the myocardial tissue of mice in the CVB3 group compared with the control group, and it was significantly upregulated in the CVB3 + Dapagroup compared with the CVB3 group (Fig. 4b).

To further investigate whether dapagliflozin alleviates myocarditis by increasing stat3 phosphorylation, we injected STATTIC into the abdominal cavity of mice with viral myocarditis. Compared with the CVB3 + Dapa group, the body weight and survival rate of the CVB3 + Dapa + STATTIC group were a little bit decreased though without significance (Fig. 3a, b). What’s more, the LVFS and LVEF of STATTIC-treated myocarditis mice were significantly reduced, indicating that ventricular function worsened after STATTIC treatment (Table 2). Besides, HE and Masson stain showed more severe inflammation and fibrosis after STATTIC-treated (Fig. 3c, d, e, f,). Consistent with this, the serum cTnI activities and IL-1β were also increased in the mice treated with STATTIC (Fig. 3g, i). However, the level of IL-6 and TNF-α did not show a similar change (Fig. 3h, j). In addition, STATTIC increased the number of CD68 + macrophages and reduced CD163 + macrophages in myocardium (3 k). Furthermore, we found that STATTIC can eliminate the upregulation of p-STAT3 induced by dapagliflozin (Fig. 4e, f), while it can also eliminate the downregulation of iNOS and IL-6 induced by dapagliflozin (Fig. 4e, g, h). These data illustrated that downregulation of activated STAT3 can significantly aggravate the severity of CVB3-induced myocarditis, eliminating the therapeutic effect of dapagliflozin, which indicates that dapagliflozin alleviate myocarditis through activating stat3 signal pathway.