Background: We recently developed novel matrix metalloproteinase-2 (MMP-2) inhibitor small molecules for cardioprotection against ischemia/reperfusion injury and validated their efficacy in ischemia/reperfusion injury in cardiac myocytes. The aim of the present study was to test our lead compounds for cardioprotection in vivo in a rat model of acute myocardial infarction (AMI) in the presence or absence of hypercholesterolemia, one of the major comorbidities affecting cardioprotection. Methods: Normocholesterolemic adult male Wistar rats were subjected to 30 min of coronary occlusion followed by 120 min of reperfusion to induce AMI. MMP inhibitors (MMPI)-1154 and -1260 at 0.3, 1, and 3 µmol/kg, MMPI-1248 at 1, 3, and 10 µmol/kg were administered at the 25th min of ischemia intravenously. In separate groups, hypercholesterolemia was induced by a 12-week diet (2% cholesterol, 0.25% cholic acid), then the rats were subjected to the same AMI protocol and single doses of the MMPIs that showed the most efficacy in normocholesterolemic animals were tested in the hypercholesterolemic animals. Infarct size/area at risk was assessed at the end of reperfusion in all groups by standard Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining, and myocardial microvascular obstruction (MVO) was determined by thioflavine-S staining. Results: MMPI-1154 at 1 µmol/kg, MMPI-1260 at 3 µmol/kg and ischemic preconditioning (IPC) as the positive control reduced infarct size significantly; however, this effect was not seen in hypercholesterolemic animals. MVO in hypercholesterolemic animals decreased by IPC only. Conclusions: This is the first demonstration that MMPI-1154 and MMPI-1260 showed a dose-dependent infarct size reduction in an in vivo rat AMI model; however, single doses that showed the most efficacy in normocholesterolemic animals were abolished by hypercholesterolemia. The further development of these promising cardioprotective MMPIs should be continued with different dose ranges in the study of hypercholesterolemia and other comorbidities.

The objective of our present study is to develop novel inhibitors for MMP-2 for acute cardioprotection. In a series of pilot studies, novel substituted carboxylic acid derivatives were synthesized based on imidazole and thiazole scaffolds and then tested in a screeening cascade for MMP inhibition. We found that the MMP-inhibiting effects of imidazole and thiazole carboxylic acid-based compounds are superior in efficacy in comparison to the conventional hydroxamic acid derivatives of the same molecules. Based on these results, a 568-membered focused library of imidazole and thiazole compounds was generated in silico and then the library members were docked to the 3D model of MMP-2 followed by an in vitro medium throughput screening (MTS) based on a fluorescent assay employing MMP-2 catalytic domain. Altogether 45 compounds showed a docking score of >70, from which 30 compounds were successfully synthesized. Based on the MMP-2 inhibitory tests using gelatin zymography, 7 compounds were then selected and tested in neonatal rat cardiac myocytes subjected to simulated I/R injury. Six compounds showed significant cardio-cytoprotecion and the most effective compound (MMPI-1154) significantly decreased infarct size when applied at 1 μM in an ex vivo model for acute myocardial infarction. This is the first demonstration that imidazole and thiazole carboxylic acid-based compounds are more efficacious MMP-2 inhibitor than their hydroxamic acid derivatives. MMPI-1154 is a promising novel cardio-cytoprotective imidazole-carboxylic acid MMP-2 inhibitor lead candidate for the treatment of acute myocardial infarction.

Purpose Matrix metalloproteinases (MMPs) are identified as modulators of the extracellular matrix in heart failure progression. However, evidence for intracellular effects of MMPs is emerging. Pro- and anti-hypertrophic cardiac effects are described. This may be due to the various sources of different MMPs in the heart tissue. Therefore, the aim of the present study was to determine the role of MMPs in hypertrophic growth of isolated rat ventricular cardiac myocytes. Methods Cardiomyocytes were isolated form ventricular tissues of the rat hearts by collagenase perfusion. RT-qPCR, western blots, and zymography were used for expression and MMP activity analysis. Cross-sectional area and the rate of protein synthesis were determined as parameters for hypertrophic growth. Results MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 mRNAs were detected in cardiomyocytes, and protein expression of MMP-2, MMP-9, and MMP-14 was identified. Hypertrophic stimulation of cardiomyocytes did not enhance, but interestingly decreased expression of MMPs, indicating that downregulation of MMPs may promote hypertrophic growth. Indeed, the nonselective MMP inhibitors TAPI-0 or TIMP2 and the MMP-2-selective ARP-100 enhanced hypertrophic growth. Furthermore, TAPI-0 increased phosphorylation and thus activation of extracellular signaling kinase (ERK) and Akt (protein kinase B), as well as inhibition of glycogen synthase 3β (GSK3β). Abrogation of MEK/ERK- or phosphatidylinositol-3-kinase(PI3K)/Akt/GSK3β-signaling with PD98059 or LY290042, respectively, inhibited hypertrophic growth under TAPI-0. Conclusion MMPs’ inhibition promotes hypertrophic growth in cardiomyocytes in vitro. Therefore, MMPs in the healthy heart may be important players to repress cardiac hypertrophy.

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