Inflammatory Bowel Disease Increases the Severity of Myocardial Infarction after Acute Ischemia-Reperfusion Injury in Mice

Abstract

(1) Background: Increased risk of myocardial infarction (MI) has been linked to several inflammatory conditions, including inflammatory bowel disease (IBD). However, the relationship between IBD and MI remains unclear. Here, we implemented an original mouse model combining IBD and MI to determine IBD's impact on MI severity and the link between the two diseases. (2) Methods: An IBD model was established by dextran sulfate sodium (DSS) administration in drinking water, alone or with oral C. albicans (Ca) gavage. IBD severity was assessed by clinical/histological scores and intestinal/systemic inflammatory biomarker measurement. Mice were subjected to myocardial ischemia-reperfusion (IR), and MI severity was assessed by quantifying infarct size (IS) and serum cardiac troponin I (cTnI) levels. (3) Results: IBD mice exhibited elevated fecal lipocalin 2 (Lcn2) and IL-6 levels. DSS mice exhibited almost two-fold increase in IS compared to controls, with serum cTnI levels strongly correlated with IS. Ca inoculation tended to worsen DSS-induced systemic inflammation and IR injury, an observation which is not statistically significant. (4) Conclusions: This is the first proof-of-concept study demonstrating the impact of IBD on MI severity and suggesting mechanistic aspects involved in the IBD-MI connection. Our findings could pave the way for MI therapeutic approaches based on identified IBD-induced inflammatory mediators.

Keywords: animal model; cytokines; inflammatory bowel disease; ischemia–reperfusion injury; myocardial infarction; translational medicine.

Figure 1

Protocol to examine the relationship between inflammatory bowel disease and severity of myocardial infarction. Chemically induced colitis in mice will be established by administration of DSS in drinking water. In addition, mice will be gavaged by C. albicans (Ca) to mimic intestinal dysbiosis in humans. Control group as well as Ca group will receive only water instead of DSS and control and DSS groups will be gavaged with vehicle PBS instead of C. albicans suspension. Stool samples and blood will be collected during treatment period to assess disease evolution. GI tract organs as well as the hearts will be harvested at the end of treatment for histological studies as well as IS measurement.

Figure 2

Experimental design of the study. Chemically induced inflammatory bowel disease (IBD) was performed by adding dextran sulfate sodium (DSS, 1% (w/v)) to drinking water from day 0 to day 9 to induce colitis, and DSS was removed on day 9 to allow remission until day 17 (DSS group). A second group of healthy mice, given only water, was used as control (Ctrl). A third group of mice was orally gavaged on day 0 with 200 µL of PBS containing 5 × 10⁷ C. albicans live cells without any other treatment (Ca). A fourth group was treated with DSS and orally gavaged with C. albicans (DSS + Ca). All animals underwent 30 min ischemia followed by either 180 min or 24 h reperfusion for IS or cTnI serum level determination, respectively. The presence of yeast in the intestinal tract was identified in the stool samples collected from each animal. On day 17, animals were either sacrificed after blood collection or subjected to ischemia–reperfusion (IR) injury (30 min of coronary occlusion followed by 180 min or 24 h of reperfusion for IS or serum cTnI level determination, respectively). The colon was removed and colon length was measured, then the different anatomical sections of the GI tract were stored in 10% buffered formalin until use for histological analysis.

Figure 3

Disease activity index evolution. Animals were monitored daily for weight loss, stool consistency, and bleeding. (A) Disease activity index (DAI) scores were attributed based on body weight loss, fecal consistency, and hematochezia. DSS administration was stopped on day 9 (black arrow) and replaced with regular water and disease evolution continued to be monitored until sacrifice of animals on day 17 (highlighted with red oval, linking time point at 17 days with panel 3C, red arrow). (B) DAI area under the curve (AUC) analysis showed a significant difference between DSS-treated and control groups. (C) DAI on day 17 showing significant remission of IBD mice. **, p < 0.01 and ***, p < 0.001 vs. control (Ctrl) group; ##, p < 0.01, ###, p < 0.001 vs. C. albicans group (Ca); $$, p < 0.01 vs. corresponding group on day 9.

Figure 4

Histological analysis of the gastrointestinal tract in DSS-treated animals. (A) Histopathology scores were determined in each organ or section of the gastrointestinal tract. (B) Overall gastrointestinal tract inflammation score was assigned to each experimental group. (C) Representative H&E-stained colon section (magnification 40×) images. (D) Representative images of the colon on day 17. Colon length was measured in the different experimental groups. *, p < 0.05, **, p < 0.01, ***, p < 0.001 vs. control (Ctrl) group; #, p < 0.05, ##, p < 0.01, ###, p < 0.001 vs. Ca group. $$$, p < 0.001, vs. corresponding DSS group.

Figure 5

Candida albicans colonization analysis. (A) Quantification of C. albicans throughout the duration of the experiment in the different groups. Stool samples were homogenized in PBS and then plated onto solid YPD medium in Petri dishes and incubated at 30 °C for 48 h. Ca colonies were counted and colonization was determined as colony-forming units per gram of stool (CFU/g). (B) Representative PAS-stained colon sections (magnification 10×, left panel; and 40×, right panel) images showing invasions and colonization of GI walls by C. albicans with different morphologies (yeast and hyphae).

Figure 6

Local inflammatory marker fecal lipocalin 2 (Lcn2) analysis by ELISA assay. Tests were performed on suspensions of stool samples diluted to 1/10 for the control and Ca groups and to 1/50 and 1/100 for the DSS groups. The graph was generated as the log₁₀ of the concentration of each group on the day of sampling. *, p < 0.05, **, p < 0.01, ***, p < 0.001 vs. corresponding control (Ctrl) group; #, p < 0.05, ##, p < 0.01, ###, p < 0.001 vs. corresponding Ca group.

Figure 7

Systemic inflammatory cytokine analysis by flow cytometry. Graphs were generated as log₁₀ of each group’s concentration. (A) Interleukin-6 (IL-6) expression levels in the different experimental groups. (B) Representative flow cytometry plots showing the variable cytokine distribution in DSS-treated group. (C) Tumor necrosis factor alpha (TNF-α) expression levels in the different experimental groups. (D) Interferon gamma (IFNγ) expression levels in the different experimental groups. (E) Interleukin-17 (IL-17) expression levels in the different experimental groups. (F) Interleukin-4 (IL-4) expression levels in the different experimental groups. (G) Interleukin-2 (IL-2) expression levels in the different experimental groups. *, p < 0.05, **, p < 0.01, ***, p < 0.001 vs. corresponding control (Ctrl) group; ##, p < 0.01, ###, p < 0.001 vs. corresponding C. albicans (Ca) group; $, p < 0.05 vs. corresponding DSS group; £, p < 0.05 vs. corresponding group on day 9.

Figure 8

Assessment of IR severity in IBD mice. IR was determined by TTC staining expressed as percentage of area at risk (IS/AAR) or cardiac troponin I (cTnI) serum level quantification by ELISA, after 30 min ischemia followed by 3 or 24-h reperfusion, respectively. (A) Heart rate (HR) before coronary occlusion, during ischemia, and after reperfusion in the different experimental groups. (B) The area at risk (AAR) was determined after 30 min of ischemia followed by 3 h of reperfusion by intravenous injection of Evans blue solution. (C) Assessment of infarct size (IS) as a percentage of the left ventricle (IS/LV %). (D) Assessment of IS as a percentage of AAR (IS/AAR %). (E) Measurement of cTnI serum levels in mice with or without treatment (DSS/Ca). The graph was generated as the log₁₀ of the concentration of each group. (F) Correlation between IR (IS/AR) and cTnI serum levels in the four experimental groups after IR (r² = 0.9554, p < 0.05). (G) Planimetry of myocardial IS. Digital photographs of midventricular slices after Evans blue and triphenyl tetrazolium chloride (TTC) staining. Infarcted areas appear pale, viable myocardium within the AAR is stained brick red. *, p < 0.05, **, p < 0.01 vs. control (Ctrl) group; #, p < 0.05 vs. C. albicans (Ca) group.

Figure 9

DSS effect on cardiomyocyte viability in vitro. Human induced pluripotent stem cell (HiPSC)-derived cardiomyocytes (HiPSC-CMs) were incubated with DSS at increasing concentrations (15, 30, and 50 µg/mL) for 48 h and cell viability was assessed by flow cytometry. Cells were tested in triplicate and results expressed as mean ± SEM for each concentration.

Figure 10

IBD–MI relationship model in mice. (A) Colitis in mice was induced by oral treatment with 1% (w/v) DSS for 9 days followed by an 8-day remission period, as evidenced by disease activity index (DAI). On day 17, myocardial ischemia–reperfusion (IR) was performed in IBD mice, while (B) local [histology (magnification 40×), colon length, and fecal lipocalin 2 (Lcn2)] and (C) systemic (IL-6) inflammation remained higher than in the control group, allowing (D) the study of the impact of IBD on the severity of MI (IS/AAR%). *, p < 0.05, **, p < 0.01, ***, p < 0.001 vs. corresponding control (Ctrl) group; #, p < 0.05, ##, p < 0.01, ###, p < 0.001 vs. corresponding Ca group; $$, p < 0.01 vs. corresponding group on day 9.