The Post-Infarction Healing Process In A Mechanically Unloaded Heart Model

Objectives: There is a growing body of evidence shown that intra-ventricular pressure and volume load play an important role in the healing process after myocardial infarction. Elevated pressure and volume load will lead to infarct expansion and ventricular dilation, even cardiac rupture. On the other hand, decreased pressure and volume load could facilitate post-infarct healing process. However, the effect of completely mechanical unloading, on the wound repair process after myocardial infarction, has not been investigated. In addition, there is no quantitative method concerning the two-dimensional distribution of collagen fibers in the infarct area. Therefore, the second purpose of the present work is to establish a novel method to accomplish this goal.Materials and Methods: We performed an isogenic Lewis-to-Lewis abdominal heterotopic heart transplantation, with the left coronary anterior descending artery (LAD) of donor heart ligated simultaneously. This rat model is characterized by completely mechanical unloading of the donor left ventricle, and leaves the infarct area healing without any influence of pressure or volume load. On the 3^rd, 7^th, 14^th and 35^th days after operation, some animals were sacrificed and the donor hearts were prepared for histological analysis and protein extraction. In situ myocardial infarction was induced by LAD ligation in Lewis rats, and the hearts were harvested at same time interval, to compare the healing process under different loading status. Collagen deposition, maturation was determined by picrosirius-red stained heart tissue sections, under circularly polarized light. Automatic angular detection of collagen fiber in the infarct area was performed by Continuity, a professional software written by the department of bioengineering, University of California. Angular data derived fromContinuity were further analyzed by circular statistics, to screening for useful parameters that were correlated with the dynamic change of two-dimensional collagen distribution in the infarct. Arteriogensis in the infarct area was evaluated by immunofluorescence. FITC-labeled wheat germ agglutinin staining was performed for determination of cross-sectional area of viable cardiomyocytes in the septum. Gelatin zymography was used for determination of matrix metalloproteinase-2 (MMP-2) and MMP-9 activity in the infarct area. The protein abundance of MMP-2, MMP-9, transforming growth factorβ1 (TGF-β1) and stromal derived factor-1 (SDF-1) was determined by western blot.Results: By performing automatic collagen fiber angle detection followed by circular statistics of the images from infarct area, three parameters concerning dispersion tendency of circular distribution (circular variance, circular standard deviation and circular dispersion) were negatively correlated with the time after myocardial infarction. Two parameters concerning concentration tendency of circular distribution (mean resultant length and von Wiese concentration) were positively correlated with the time after myocardial infarction. These five parameters thus could quantitatively estimate the dynamic change of two-dimensional distribution of collagen fiber in the infarct. As time went on, there was an increasing trend for collagen fiber disorganization in the infarct area of the unloaded hearts. This process could be quantitatively estimated by the above five parameters. The unloaded infarct area exhibited decreased level of collagen deposition and maturation, compared with in situ infarct group. The arteriolar density in the infarct area was high in unloaded hearts from 14^th day after operation. There was a continuously decreasing of heart weight and cross-sectional area, and increased level of collagen deposition in non-infarct area of mechanical unloaded hearts. Compared with in situ group, there is a great amount of inflammatory cells infiltration in the unloaded infarct area, and this phenomenon was coupled with increased level and activity of MMP-9, and increased abundance of TGF-β1 protein. These were no significant changes of MMP-2 and SDF-1 protein level between in situ infarct group and unloading group at the same time points.Conclusions: Automatic angular detection combined with circular statistics is a novel quantitative method to estimate two-dimensional distribution of collagen fiber organization in the infarct area. The infarct healing process in completely unloaded heart is characterized by decreased collagen deposition and maturation, and an increasing trend for collagen fiber disorganization, with delayed resolution of inflammation. Elevated level of MMP-9 and TGF-β1 in the mid-late phase of post-infarct healing, may participate in this abnormal wound repair process. Thus, these results suggest that abdominal heterotopic transplantation of infarcted heart could be a useful model to investigate the mechanisms of delayed healing process after myocardial infarction.