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. 2022 Feb;600(3):547-567.
doi: 10.1113/JP282153. Epub 2021 Dec 13.

Metabolic design in a mammalian model of extreme metabolism, the North American least shrew (Cryptotis parva)

Dillon J Chung  1 , Grey P Madison  1 , Angel M Aponte  1 , Komudi Singh  1 , Yuesheng Li  1 , Mehdi Pirooznia  1 , Christopher K E Bleck  1 , Nissar A Darmani  2 , Robert S Balaban  1
Affiliations

Metabolic design in a mammalian model of extreme metabolism, the North American least shrew (Cryptotis parva)

Dillon J Chung et al. J Physiol. 2022 Feb.

Abstract

Mitochondrial adaptations are fundamental to differentiated function and energetic homeostasis in mammalian cells. But the mechanisms that underlie these relationships remain poorly understood. Here, we investigated organ-specific mitochondrial morphology, connectivity and protein composition in a model of extreme mammalian metabolism, the least shrew (Cryptotis parva). This was achieved through a combination of high-resolution 3D focused ion beam electron microscopy imaging and tandem mass tag mass spectrometry proteomics. We demonstrate that liver and kidney mitochondrial content are equivalent to the heart, permitting assessment of mitochondrial adaptations in different organs with similar metabolic demand. Muscle mitochondrial networks (cardiac and skeletal) are extensive, with a high incidence of nanotunnels - which collectively support the metabolism of large muscle cells. Mitochondrial networks were not detected in the liver and kidney as individual mitochondria are localized with sites of ATP consumption. This configuration is not observed in striated muscle, likely due to a homogeneous ATPase distribution and the structural requirements of contraction. These results demonstrate distinct, fundamental mitochondrial structural adaptations for similar metabolic demand that are dependent on the topology of energy utilization process in a mammalian model of extreme metabolism. KEY POINTS: Least shrews were studied to explore the relationship between metabolic function, mitochondrial morphology and protein content in different tissues. Liver and kidney mitochondrial content and enzymatic activity approaches that of the heart, indicating similar metabolic demand among tissues that contribute to basal and maximum metabolism. This allows an examination of mitochondrial structure and composition in tissues with similar maximum metabolic demands. Mitochondrial networks only occur in striated muscle. In contrast, the liver and kidney maintain individual mitochondria with limited reticulation. Muscle mitochondrial reticulation is the result of dense ATPase activity and cell-spanning myofibrils which require networking for adequate metabolic support. In contrast, liver and kidney ATPase activity is localized to the endoplasmic reticulum and basolateral membrane, respectively, generating a locally balanced energy conversion and utilization. Mitochondrial morphology is not driven by maximum metabolic demand, but by the cytosolic distribution of energy-utilizing systems set by the functions of the tissue.

Keywords: allometry; inter-organelle interactions; metabolism; mitochondria; reticulum.

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Conflict of interest statement

Competing interests

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.. Representative segmentation of mitochondria from four tissues in Cryptotis parva
Data are raw FIB-SEM images (greyscale) with an overlay of mitochondrial segmentation (false colour) from heart (A), skeletal muscle (B), liver (C) and kidney (D). Distinct colours indicate individually segmented mitochondria. Scale bar: 5 μm. [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 2.
Figure 2.. Morphological characteristics of mitochondria and mitochondrial networks in Cryptotis parva
A, mitochondrial volume density. B, mitochondrial complexity index – an estimate of networking structure. C, individual mitochondrial volume. D, mitochondrial sphericity. E, mitochondrial surface area to volume ratio. Numbered asterisks are cell identifiers demonstrating the increase in estimated mitochondrial volume density associated with greater sampling of paravascular and paranuclear skeletal muscle mitochondria. Different letters indicate a significant difference among tissues (ANOVA, P < 0.0001). Crosses indicate a significant difference between mitochondria and networked mitochondria within a muscle type (Mann–Whitney, P < 0.0001). Asterisks indicate a significant difference between skeletal muscle and cardiac mitochondrial networks (Mann–Whitney, P < 0.0001). Skeletal muscle, n = 19,788 mitochondria, 14 datasets, 3 animals; cardiac muscle, n = 15,893 mitochondria, 18 datasets, 3 animals; kidney, n = 16,143 mitochondria, 6 datasets, 2 animals; liver, n = 6939 mitochondria, 13 datasets, 3 animals. Skeletal muscle networks, n = 242 networks, 16 datasets, 3 animals; cardiac muscle, n = 1218 networks, 18 datasets, 3 animals. Data are means ± SD and exclude values associated with numbered asterisks when present.
Figure 3.
Figure 3.. Cryptotis parva exhibits high mitochondrial connectivity
Intermitochondrial junctions (IMJs, arrows) are prevalent in the heart (A–D) and skeletal muscle (E–H). Some skeletal muscle intermyofibrillar IMJs form unique structures with high outer mitochondrial membrane surface area – termed mitosynapses (I–L, asterisks). Data are TEM images; scale bar: 500 nm. M, mean volume density of IMJ-linked mitochondrial networks. Note the increase in volume density between individual mitochondria (N) and IMJ-linked mitochondrial networks (O). Asterisks indicate a significant difference between muscle type (Tukey’s or Mann–Whitney’s test, P < 0.0001). Summary data are means ± SD. Skeletal muscle networks, n = 682 networks, 14 datasets, 3 animals; cardiac muscle, n = 1218 networks, 18 datasets, 3 animals.
Figure 4.
Figure 4.. Mitochondrial networks are limited to striated muscle and recruit nanotunnels as part of their structure
3D renderings of a single mitochondrial network in skeletal muscle (A–B) and heart (C–D), oriented parallel (A, C) or perpendicular (B, D) to the plane of contraction (double arrow). Mitochondrial networks are not observed in the kidney (E) or liver (F) and mitochondria are distributed throughout the cytosol in these tissues. Distinct colours indicate individual mitochondria and 3D renderings are partially overlaid on FIB-SEM volumes. I, mitochondrial nanotunnel frequency. J, nanotunnel diameter. K, nanotunnel length. Asterisks indicate a significant difference between tissues (t test P = 0.0007 or Mann–Whitney’s test, P < 0.0001). Summary data are means ± SD. Skeletal muscle, n = 100 nanotunnels, 8 datasets, 2 animals; cardiac muscle, n = 286 nanotunnels, 8 datasets, 3 animals. [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 5.
Figure 5.. Liver mitochondria exhibit extensive inter-organelle interactions
A, frequency distribution of individual mitochondrial surface area (percentage of total) within 50 nm of the ER – the minimum distance required to detect a significant interaction between these organelles (subset figure, data are means ± SD; asterisks indicate significant difference from 0 nm, Tukey’s test, P = 0.0068). B, individual mitochondrial volume is positively correlated with mitochondrial-associated ER surface area (simple linear regression, y = 1.56x + 0.87, R2 = 0.64, P < 0.0001; n = 8941 mitochondria, 3 animals). C–H, liver mitochondria exhibit membrane contacts across the plasma membrane (PM; paracellular membrane contact, PcMC; black arrow). PcMC formation involves (1) mitochondria aligning at the PM and deformation of the outer mitochondrial membrane (I–J), (2) recruitment of ER between mitochondrial membranes and the PM (K–L), and (3) formation of the PcMC (M–N). Data are TEM images, scale bar: 500 μm unless otherwise indicated.
Figure 6.
Figure 6.. Liver paracellular mitochondrial contacts (PcMCs) are not loose associations
Poor sample fixation of least shrew (Cryptotis parva) liver results in delamination of adjacent hepatocyte plasma membranes. Despite poor fixation, PcMCs remain intact, indicating that a structure holds these mitochondria in place. Data are raw TEM images. Scale bar: 500 nm unless otherwise indicated.
Figure 7.
Figure 7.. Liver and kidney mitochondrial protein programming approaches that of the heart in Cryptotis parva
A, whole-tissue cytochrome a content. B, whole-tissue cytochrome c content. Data are means ± SD and different letters indicate a significant difference between tissues (ANOVA, P < 0.0001, n = 7 animals). C–E, heart normalized protein abundance for biochemical pathways associated with mitochondria. Individual protein subunit abundance (datapoints) can be found in Data S1. Dashed lines indicate mean abundance value within a tissue. n = 4 animals.
Figure 8.
Figure 8.. Least shrew (Cryptotis parva) skeletal muscle intermyofibrillar mitochondria have a high cristae surface area
Data are raw TEM images. Scale bar: 500 nm.
See this image and copyright information in PMC

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