Molecular screening in a translational large animal trial identifies a differential inflammatory response for MINOCA

Figures

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  Fig. 1. Schematic illustration of the animal model and sampling strategy. To mimic MINOCA, a carotid crush maneuver was performed to generate microthrombi which were subsequently injected into the coronary artery to induce CME. To reduce confounders, MI animals also received carotid crush, but no microthrombi were injected. MI animals were subjected to 90 min of coronary artery balloon occlusion followed by reperfusion. After MINOCA/MI was induced, plasma samples were collected at 150 and 300 min post-procedure. Following euthanasia, a high-resolution MRI was performed in addition to extensive tissue sampling for histology and downstream molecular assays

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  Fig. 2. CME-derived MINOCA and MI display significantly different infarction sizes accompanied by cell death and interstitial hemorrhage. a Troponin and CK kinetics following infarction induction in both, MI and MINOCA animals. N = 7 for MI and MINOCA. b Percentage of scar tissue of total ventricular mass as calculated by ex-vivo LGE CMR in both, MI and MINOCA animals. White arrows in MRI images indicate areas of infarction. N = 6 for MI, N = 7 for MINOCA. c Percent tissue necrosis in AHA heart segments affected either by MI or MINOCA, as evaluated by histology of tissues in the infarction area. Scale bar = 4 mm. N = 7 for MI and MINOCA. d Frequency of interstitial hemorrhage in the infarction area as assessed by H&E in the infarction area of either MI or MINOCA animals. N = 7 for MI and MINOCA. Arrows denote specific areas of interest. Data are displayed as box and whisker plots with individual points. Boxplots display the 95% confidence interval. Scale bar = 400 µm. Statistical significance for (a) was determined using a two-way ANOVA comparing the two models at their respective timepoints. Statistical significance for (b), (c), and (d) was determined by a two-tailed unpaired T-test…

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  Fig. 3. MINOCA and MI display significantly different inflammatory cell infiltration patterns in the infarction area. a Graphical overview on different sections assessed during histology via H&E staining. b Leukocyte infiltrates in the supply area of the coronary artery undergoing either balloon occlusion (MI) or microthrombus injection (MINOCA) were quantified in tissue samples. Arrows mark leukocyte infiltrates. Scale bar: 400 µm. Statistical significance was determined via a Brown–Forsythe and Welch ANOVA with a Dunnett’s T3 multiple comparisons test. c Leukocyte infiltrates in specific areas affected by microthrombosis were quantified in various regions of MINOCA animals, starting with the thrombus area (TA), the perivascular area (PA), and the thrombus-associated area (TAA). Arrows denote specific areas of interest. Scale bar for TA and PA: 200 µm. Scale bar for TAA: 400 µm. Data are displayed as box and whisker plots with individual points. Boxplots display the 95% confidence interval. Statistical significance was determined via a Brown–Forsythe and Welch ANOVA with a Dunnett’s T3 multiple comparisons test. Asterisks indicate p values: **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, only…

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  Fig. 4. MINOCA and MI induce prominent inflammatory responses with lead cytokine differences. Serum samples were collected 0, 150, and 300 min after infarction induction in both MI (red) and MINOCA (blue) animals were collected and subsequently pro-inflammatory and anti-inflammatory cytokine expression were assessed using multi-ELISA. a Cytokines with similar temporal kinetics of both, MI and MINOCA animals. b Significantly different cytokines in samples of both, MI and MINOCA animals across sampling timepoints. Data are displayed as mean ± SEM. Asterisks indicate p-values: *p ≤ 0.05, **p ≤ 0.01 (comparison of disease models at a given timepoint); #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.001 (comparison of MINOCA across timepoints); $p ≤ 0.05 (comparison of MI across timepoints) with only significant values shown. N = 4 for MI and N = 6 for MINOCA. Abbreviations: MI, classic myocardial infarction; MINOCA, myocardial infarction with non-obstructive coronary arteries

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  Fig. 5. CME-derived MINOCA and MI induce distinct miRNA profiles of ischemia reperfusion injury. miRNA of plasma samples at 0, 150, and 300 min following infarction induction in both MI and MINOCA animals was isolated. Subsequently, miRNA profiling was performed. a Heatmap of altered miRNA expression levels in MI animals over time. b Heatmap of altered miRNA expression levels in MINOCA animals over time. c Principal component analysis of miRNA profiles in MI and MINOCA animals over time. miRNAs with highest FDR and fold change are highlighted for both MI and MINOCA animals. d Differential expression analysis comparing MI and MINOCA depicted in a volcano plot. Significantly different expressed miRNAs are colored. N = 6 for MI and MINOCA plasma taken at 0 min and 150 min. N = 5 for MI and MINOCA plasma taken at 300 min. Statistical significance was determined by FDR ≤ 0.1, Log2FC ≥ 1.5, and Log2FC ≤ − 1.5. Abbreviations: MI, classic myocardial infarction; MINOCA, myocardial infarction with non-obstructive coronary arteries

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  Fig. 6. Leukotriene signaling constitutes a inflammatory pathway underlying MINOCA. a–d Plasma samples were collected at 0, 150, and 300 min following infarction induction in both MI and MINOCA animals. Subsequently, a proteomic analysis was performed. a Heatmap of significantly altered protein expression levels in MI animals over time. The heatmap scale bar represents the mean normalized protein abundance measured via LC/MS, with high mean normalized abundance in blue and low mean normalized abundance in orange. b Heatmap of significantly altered protein expression levels in MINOCA animals over time. The heatmap scale bar represents the mean normalized protein abundance measured via LC/MS, with high mean normalized abundance in blue and low mean normalized abundance in orange. c Differential expression analysis displaying significantly differently expressed proteins between MINOCA and MI at 300 min in a volcano plot. Proteins involved in anaerobic glycolysis are highlighted for MI animals, while leukotriene A4 hydrolase is highlighted for MINOCA animals. N = 6 for MINOCA and MI. Statistical significance was determined by FDR ≤ 0.1, Log2FC ≥ 1.5, and Log2FC ≤ -1.5. d Gene ontology…