Comparison of myocardial remodeling between cryoinfarction and reperfused infarction in mice

Abstract

Myocardial infarction is associated with inflammatory reaction leading to tissue remodeling. We compared tissue remodeling between cryoinfarction (cMI) and reperfused myocardial infarction (MI) in order to better understand the local environment where we apply cell therapies. Models of closed-chest one-hour ischemia/reperfusion MI and cMI were used in C57/Bl6-mice. The reperfused MI showed rapid development of granulation tissue and compacted scar formation after 7 days. In contrast, cMI hearts showed persistent cardiomyocyte debris and cellular infiltration after 7 days and partially compacted scar formation accompanied by persistent macrophages and myofibroblasts after 14 days. The mRNA of proinflammatory mediators was transiently induced in MI and persistently upregulated in cMI. Tenascin C and osteopontin-1 showed delayed induction in cMI. In conclusion, the cryoinfarction was associated with prolonged inflammation and active myocardial remodeling when compared to the reperfused MI. These substantial differences in remodeling may influence cellular engraftment and should be considered in cell therapy studies.

Figure 1

Comparison of myocardial histopathology between cryoinfarction and reperfused infarction. Hematoxylin-eosin staining of left ventricular lateral wall in cryoinfarction after (a) 3 days an increased cellular infiltration with extensive cardiomyocyte debris, with (b) formation of granulation tissue after 7 days, and (c) an almost compacted scar formation after 14 days postinjury with permanent occlusion of a large coronary vessel (arrow). In parts (a)-(c), a higher magnification (400x) insert shows cellular and matrix content in detail. In contrast, (d) reperfused myocardial infarction shows granulation tissue formation after 3 days and (e) a compacted, nontransmural scar formation after 7 days reperfusion. Picrosirius red staining of (f) cryoinfarction after 7 days reveals loose collagen fibers, while (g) reperfused infarction presents a compacted, collagen-rich scar. Alpha smooth muscle actin staining of myofibroblasts (black-stained cells) indicates (h) active interstitial remodeling 7 days after cryoinfarction whereas (i) reperfused infarction has only few positive cells in the scar (eosin counterstaining). Scale bar: 150 μm.

Figure 2

Neutrophils density in injured myocardium. Representative MCA 771 G staining for neutrophils (black-stained cells) in infarcted left ventricular wall (pink eosin counterstaining) 3 days after (a) reperfused infarction (MI) and (b) cryoinfarction (cMI). Comparison of neutrophils cell density in (c) infarcted area and (d) noninfarcted area in both models. Scale bar: 75 μm; *P < .05.

Figure 3

Macrophage density in cryoinfarction and reperfused infarction. Representative F4/80 staining for macrophages (black-stained cells) in left ventricular wall (pink eosin counterstaining) 7 days after (a) reperfused infarction (MI) and (b) cryoinfarction (cMI). Elastase staining showed lack of macrophage activity after 7 days in (c) MI in contrast to (d) ongoing activity (black staining) in cMI (pink eosin counterstaining). Comparison of macrophage cell density in (e) infarcted area and (f) noninfarcted area in both models. Scale bar (a)-(b): 75 μm, (c)-(d): 100 μm; *P < .05.

Figure 4

Osteopontin-1 in infarcted myocardium. Representative staining for macrophage maturation marker osteopontin-1 (black-stained cells) in left ventricular wall (eosin counterstaining) 7 days after (a) reperfused infarction (MI) and (b) cryoinfarction (cMI). Cell density of osteopontin-1-positive cells in (c) infarcted area and (d) noninfarcted area in both models. Scale bar: 75 μm; *P < .05.

Figure 5

Expression profile of inflammatory mediators in cryoinfarction and reperfused infarction. RT-qPCR-analysis of mRNA-expression of (a) free radicals scavenger enzyme heme oxygenase 1, (b) proinflammatory cytokine TNF-α, (c) anti-inflammatory cytokine IL-10, (d) macrophage-related chemokine CCL2, (e) neutrophils-related chemokine CCL3 and (f) macrophage-related chemokine CCL4 in cryoinfarction (cMI) and reperfused infarction (MI). Data are normalized to respective shams and housekeeping gene GAPDH. *P < .05.

Figure 6

Expression profile of remodeling-related mediators in cryoinfarction and reperfused infarction. RT-qPCR-analysis of mRNA-expression of (a) macrophage maturation marker osteopontin-1, (b) early remodeling marker tenascin C, and isoforms of TGF-β, an anti-inflammatory cytokine and remodeling mediator (c) TGF-β1, (d) TGF- β2, and (e) TGF-β3 in cryoinfarction (cMI) and reperfused infarction (MI). Data are normalized to respective shams and housekeeping gene GAPDH. *P < .05.