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Changes in myocardial microstructure are important components of the tissue response to infarction but are difficult to resolve with current imaging techniques. A novel technique, diffusion spectrum MRI tractography (DSI tractography), was thus used to image myofiber architecture in normal and infarcted myocardium. Unlike diffusion tensor imaging, DSI tractography resolves multiple myofiber populations per voxel, thus generating accurate 3D tractograms, which we present in the myocardium for the first time.DSI tractography was performed at 4.7 T in excised rat hearts 3 weeks after left coronary artery ligation (n=4) and in 4 age-matched controls. Fiber architecture in the control hearts varied smoothly from endocardium to epicardium, producing a symmetrical array of crossing helical structures in which orthogonal myofibers were separated by fibers with intermediate helix angles. Fiber architecture in the infarcted hearts was severely perturbed. The infarct boundary in all cases was highly irregular and punctuated repeatedly by residual myofibers extending from within the infarct to the border zones. In all infarcts, longitudinal myofibers extending toward the basal-anterior wall and transversely oriented myofibers extending toward the septum lay in direct contact with each other, forming nodes of orthogonal myofiber intersection or contact.DSI tractography resolves 3D myofiber architecture and reveals a complex network of orthogonal myofibers within infarcted myocardium. Meshlike networks of orthogonal myofibers in infarcted myocardium may resist mechanical remodeling but also probably increase the risk for lethal reentrant arrhythmias. DSI tractography thus provides a new and important readout of tissue injury after myocardial infarction.