Person:

Hammer, Peter

Background

Previous studies have demonstrated that alterations in myocardial structure, consistent with tissue and sarcomere disruption as well as myofibril dissociation, occur after myocardial ischemia and reperfusion. In this study we determine the onset of these structural changes and their contribution to electrical conduction.

Methods

Langendorff perfused rabbit hearts (n = 47) were subjected to 0, 5, 10, 15, 20, 25, and 30 minutes global ischemia, followed by 120 minutes reperfusion. Hemodynamics were recorded and tissue samples were collected for histochemical and immunohistochemical studies. Orthogonal epicardial conduction velocities were measured, with temperature controlled, in a separate group of 10 hearts subjected to 0 or 30 minutes of global ischemia, followed by 120 minutes of reperfusion.

Results

Histochemical and quantitative light microscopy spatial analysis showed significantly increased longitudinal and transverse interfibrillar separation after 15 minutes or more of ischemia (p < 0.05 versus control). Confocal immunohistochemistry and Western blot analysis demonstrated significant reductions (p < .05 versus control) of the intercellular adherens junction protein, N-cadherin, and the active phosphorylated isoform of the principal gap junction protein, connexin 43 at more than 15 minutes of ischemia. Cellular redistribution of connexin 43 was also evidenced on immunohistochemistry. No change in integrin-β1, an extracellular matrix attachment protein, or in epicardial conduction velocity anisotropy was observed.

Conclusions

These data indicate that there are significant alterations in the structural integrity of the myocardium as well as gap and adherens junction protein expression with increasing global ischemia time. The changes occur coincident with previously observed significant decreases in postischemic functional recovery, but are not associated with altered expression of matrix binding proteins or electrical anisotropic conduction.

The purpose of this study was to determine the ability of superparamagnetic iron oxide (SPIO) nanoparticles to function as a long-term tracking label for multi-modal imaging of implanted engineered tissues containing muscle-derived progenitor cells using magnetic resonance imaging (MRI) and X-ray micro-computed tomography (μCT). SPIO-labeled primary myoblasts were embedded in fibrin sealant and imaged to obtain intensity data by MRI or radio-opacity information by μCT. Each imaging modality displayed a detection gradient that matched increasing SPIO concentrations. Labeled cells were then incorporated in fibrin sealant, injected into the atrioventricular groove of rat hearts, and imaged in vivo and ex vivo for up to 1 year. Transplanted cells were identified in intact animals and isolated hearts using both imaging modalities. MRI was better able to detect minuscule amounts of SPIO nanoparticles, while μCT more precisely identified the location of heavily-labeled cells. Histological analyses confirmed that iron oxide particles were confined to viable, skeletal muscle-derived cells in the implant at the expected location based on MRI and μCT. These analyses showed no evidence of phagocytosis of labeled cells by macrophages or release of nanoparticles from transplanted cells. In conclusion, we established that SPIO nanoparticles function as a sensitive and specific long-term label for MRI and μCT, respectively. Our findings will enable investigators interested in regenerative therapies to non-invasively and serially acquire complementary, high-resolution images of transplanted cells for one year using a single label.