Fig 1.
A schematic representation of the experimental procedures.
(A) For imaging of mitochondrial distribution, the ischemic interval was 30 minutes followed by 10 minutes of reperfusion (top). Hearts used for functional and infarction measurements were subjected to 30 minutes of ischemia followed by 2 hours of reperfusion (bottom). In both instances, mitochondria were delivered to the heart at the onset of reperfusion. (B) Cultured human cardiac fibroblasts (fluorescently stained in this phase contrast image overlay with TOMM20 [green] to show mitochondria and DAPI [blue] to show nuclei), were used to isolate and label mitochondria with 18F-R6G (colorized as red in this transmission electron micrograph) and 30 nm iron oxide particles (black dots on the mitochondrial outer surface). Dual-labeled mitochondria were injected or perfused into ischemic Langendorff-perfused isolated hearts, which were imaged by PET and μCT followed with MRI. Hearts were then fixed, embedded, sectioned, and histologically stained for fluorescence and brightfield microscopy.
Fig 2.
Transmission electron microscopy of iron oxide-labeled human mitochondria and ATP concentrations in labeled versus unlabeled isolated mitochondria.
(A) Representative low magnification images of unlabeled adult human cardiac fibroblast mitochondria (left panel) and iron oxide-labeled mitochondria (right panel). (B) Representative high magnification images of 30 nm magnetic iron oxide particles associated with the outer mitochondrial membrane are indicated with arrows. Scale bars equal 500 nm. (C) ATP concentrations in unlabeled and iron oxide nanoparticle-labeled isolated human cardiac fibroblast mitochondria (left). A high magnification image of unlabeled mitochondria is shown at the right and the scale bar equals 500 nm. Isolated mitochondria were electron dense and ≤ 1% appeared fractured or damaged, regardless of whether they were unlabeled or cross-linked to iron oxide nanoparticles.
Fig 3.
Imaging of a regionally ischemic heart injected with dual-labeled human mitochondria.
(A) μCT, PET, and merged volumetric renderings are shown from left to right. The metal suture (bright signal on μCT) indicates the site of LAD coronary artery ligation. (B) Coronal slices of MRI (0.148 mm thickness) along with the corresponding PET scan as well as the merged MRI and PET images are shown (left to right). (C) Transverse slices of MRI, PET, and the merged images (left to right). Regions of hypointense T2-weighted MRI signals from iron correlate with the PET signals from 18F-R6G.
Fig 4.
Histology of globally or regionally ischemic rabbit hearts injected with human mitochondria.
(A) Injected heart sections were fluorescently stained for the muscle markers desmin (green), the human-specific mitochondrial marker MTCO2 (red), and nuclei using the DNA stain DAPI (blue). (B) Fluorescent staining with the membrane marker WGA (red), the 113–1 human mitochondrial marker (green), and DAPI (blue). (C) MTCO2 and nuclear staining is shown with phase contrast illumination. (D) Prussian blue (blue) and pararosaniline (pink) staining of injected mitochondria labeled with magnetic iron oxide nanoparticles. Scale bars represent 25 μm. Transplanted mitochondria associated with cardiac myocyte sarcolemmata are indicated (arrows).
Fig 5.
Imaging of a regionally ischemic heart perfused with dual-labeled human mitochondria.
(A) Volumetric renderings of μCT, PET, and the merged acquisitions are shown (left to right). The μCT signal from the metal suture indicates the site of LAD coronary artery ligation. (B) Coronary slices from MRI (0.148 mm thickness) along with the corresponding PET scan as well as the merged MRI and PET images are depicted (left to right). (C) Transverse slices of MRI (0.148 mm thickness), PET, and the merged images (left to right) from the same perfused heart. Regions of hypointense T2-weighted MRI signals from iron correlate with PET signals from 18F-R6G.
Fig 6.
Histology of regionally ischemic hearts perfused with human mitochondria.
(A) Perfused heart sections were stained with the muscle marker α-actinin (red) and the human mitochondrial marker MTCO2 (green) to show the position of transplanted mitochondria in the heart (arrows). (B) Some hearts were also perfused with FITC-lectin (green) prior to fixation to display luminal surfaces of blood vessels. These sections were counter-stained with the 113–1 human mitochondrial marker (red) and nuclei were stained with DAPI (blue). The staining shows transplanted mitochondria associated with the vasculature, within interstitial spaces, and attached to cardiomyocytes. (C) Prussian blue staining (blue) and a pararosaniline counterstain (pink) confirmed transplanted mitochondria were labeled with magnetic iron oxide nanoparticles. Scale bars equal 25 μm.
Fig 7.
Myocardial function in regionally ischemic hearts perfused with autologous rabbit liver mitochondria.
(A) End diastolic pressure (mm Hg); (B) positive dP/dt (mm Hg/s x 100); (C) % segmental shortening (end-diastolic length [EDL] minus end-systolic length [ESL] over end-diastolic length [EDL] x 100) in Control and Mitochondria heart groups, pre-ischemia, and during 30 minutes regional ischemia and 120 minutes of reperfusion. Sham groups were not subjected to ischemia and reperfusion or mitochondrial treatment. (D) AAR (% left ventricular volume) and infarct size (% AAR) following 30 minutes regional ischemia and 120 minutes reperfusion in Control and Mitochondria-treated hearts (left and middle, respectively). (A-D) * indicates a p < 0.001 between the Sham group and both Control and Mitochondria groups for EDP, dP/dt, and % segmental shortening at the indicated times. ** indicates a p < 0.001 between Control and Mitochondria-treated groups for EDP, dP/dt, and % segmental shortening at the indicated times as well as for infarct size at the end of reperfusion.