The cardiolipin-binding peptide elamipretide mitigates fragmentation of cristae networks following cardiac ischemia reperfusion in rats.

Mitochondrial dysfunction contributes to cardiac pathologies. Barriers to new therapies include an incomplete understanding of underlying molecular culprits and a lack of effective mitochondria-targeted medicines. Here, we test the hypothesis that the cardiolipin-binding peptide elamipretide, a clinical-stage compound under investigation for diseases of mitochondrial dysfunction, mitigates impairments in mitochondrial structure-function observed after rat cardiac ischemia-reperfusion. Respirometry with permeabilized ventricular fibers indicates that ischemia-reperfusion induced decrements in the activity of complexes I, II, and IV are alleviated with elamipretide. Serial block face scanning electron microscopy used to create 3D reconstructions of cristae ultrastructure reveals that disease-induced fragmentation of cristae networks are improved with elamipretide. Mass spectrometry shows elamipretide did not protect against the reduction of cardiolipin concentration after ischemia-reperfusion. Finally, elamipretide improves biophysical properties of biomimetic membranes by aggregating cardiolipin. The data suggest mitochondrial structure-function are interdependent and demonstrate elamipretide targets mitochondrial membranes to sustain cristae networks and improve bioenergetic function.

Visualizing melanosomes, lipofuscin, and melanolipofuscin in human retinal pigment epithelium using serial block face scanning electron microscopy.

To assess serial section block-face scanning electron microscopy (SBFSEM) for retinal pigment epithelium (RPE) ultrastructure, we determined the number and distribution within RPE cell bodies of melanosomes (M), lipofuscin (L), and melanolipofuscin (ML). Eyes of 4 Caucasian donors (16M, 32F, 76F, 84M) with unremarkable maculas were sectioned and imaged using an SEM fitted with an in-chamber automated ultramicrotome. Aligned image stacks were generated by alternately imaging an epoxy resin block face using backscattered electrons, then removing a 125 nm-thick layer. Series of 249-499 sections containing 5-24 nuclei were examined per eye. Trained readers manually assigned boundaries of individual cells and x,y,z locations of M, L, and ML. A Density Recovery Profile was computed in three dimensions for M, L, and ML. The number of granules per RPE cell body in 16M, 32F, 76F, and 84M eyes, respectively, was 465 ± 127 (mean ± SD), 305 ± 92, 79 ± 40, and 333 ± 134 for L; 13 ± 9; 6 ± 7, 131 ± 55, and 184 ± 66 for ML; and 29 ± 19, 24 ± 12, 12 ± 7, and 7 ± 3 for M. Granule types were spatially organized, with M near apical processes. The effective radius, a sphere of decreased probability for granule occurrence, was 1 µm for L, ML, and M combined. In conclusion, SBFEM reveals that adult human RPE has hundreds of L, LF, and M and that granule spacing is regulated by granule size alone. When obtained for a larger sample, this information will enable hypothesis testing about organelle turnover and regulation in health, aging, and disease, and elucidate how RPE-specific signals are generated in clinical optical coherence tomography and autofluorescence imaging.

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy.

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer's and Parkinson's diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of <200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.