Ischemic heart disease is associated with inflammation, interstitial fibrosis and ventricular dysfunction prior to the development of heart failure. Endocannabinoids and the cannabinoid receptor CB2 have been claimed to be involved, but their potential role in cardioprotection is not well understood. We therefore explored the role of the cannabinoid receptor CB2 during the initial phase of ischemic cardiomyopathy development prior to the onset of ventricular dysfunction or infarction. Wild type and CB2-deficient mice underwent daily brief, repetitive ischemia and reperfusion (I/R) episodes leading to ischemic cardiomyopathy. The relevance of the endocannabinoid-CB2 receptor axis was underscored by the finding that CB2 was upregulated in ischemic wild type cardiomyocytes and that anandamide level was transiently increased during I/R. CB2-deficient mice showed an increased rate of apoptosis, irreversible loss of cardiomyocytes and persistent left ventricular dysfunction 60 days after the injury, whereas wild type mice presented neither morphological nor functional defects. These defects were due to lack of cardiomyocyte protection mechanisms, as CB2-deficient hearts were in contrast to controls unable to induce switch in myosin heavy chain isoforms, antioxidative enzymes and chemokine CCL2 during repetitive I/R. In addition, a prolonged inflammatory response and adverse myocardial remodeling were found in CB2-deficient hearts because of postponed activation of the M2a macrophage subpopulation. Therefore, the endocannabinoid-CB2 receptor axis plays a key role in cardioprotection during the initial phase of ischemic cardiomyopathy development.

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Vessel formation is of critical importance for organ function in the normal and diseased state. In particular, the labeling and quantitation of small vessels prove to be technically challenging using current approaches. We have, therefore, established a transgenic embryonic stem (ES) cell line and a transgenic mouse model where the vascular endothelial growth factor receptor VEGFR-1 (flt-1) promoter drives the expression of the live reporter eGFP. Fluorescence microscopy and immunostainings revealed endothelial-specific eGFP labeling of vascular networks. The expression pattern recapitulates that of the endogenous flt-1 gene, because small and large vessels are labeled by eGFP during embryonic development; after birth, the expression becomes more restricted to small vessels. We have explored this in the cardiovascular system more in detail and found that all small vessels and capillaries within the heart are strongly eGFP+. In addition, myocardial injuries have been induced in transgenic mice and prominent vascular remodeling, and an increase in endothelial cell area within the peri-infarct area could be observed underscoring the utility of this mouse model. Thus, the transgenic flt-1/eGFP models are powerful tools to investigate and quantify vascularization in vivo and to probe the effect of different compounds on vessel formation in vitro.

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Repetitive brief ischemia and reperfusion (I/R) is associated with ventricular dysfunction in pathogenesis of murine ischemic cardiomyopathy and human hibernating myocardium. We investigated the role of matricellular protein osteopontin-1 (OPN) in murine model of repetitive I/R. One 15-min LAD-occlusion followed by reperfusion was performed daily over 3, 5, and 7 consecutive days in C57/Bl6 wildtype- (WT-) and OPN−/−-mice (n = 8/group). After echocardiography hearts were processed for histological and mRNA-studies. Cardiac fibroblasts were isolated, cultured, and stimulated with TGF-β1. WT-mice showed an early, strong, and cardiomyocyte-specific osteopontin-expression leading to interstitial macrophage infiltration and consecutive fibrosis after 7 days I/R in absence of myocardial infarction. In contrast, OPN−/−-mice showed small, nontransmural infarctions after 3 days I/R associated with significantly worse ventricular dysfunction. OPN−/−-mice had different expression of myocardial contractile elements and antioxidative mediators and a lower expression of chemokines during I/R. OPN−/−-mice showed predominant collagen deposition in macrophage-rich small infarctions. We found lower induction of tenascin-C, MMP-9, MMP-12, and TIMP-1, whereas MMP-13-expression was higher in OPN−/−-mice. Cultured OPN−/−-myofibroblasts confirmed these findings. In conclusion, osteopontin seems to modulate expression of contractile elements, antioxidative mediators, and inflammatory response and subsequently remodel in order to protect cardiomyocytes in murine ischemic cardiomyopathy.