Mitochondrial Surface Area Research Articles

Increased mitochondrial surface area and cristae density in the skeletal muscle of strength athletes.

Mitochondria are the cellular organelles responsible for resynthesising the majority of ATP. In skeletal muscle, there is an increased ATP turnover during resistance exercise to sustain the energetic demands of muscle contraction. Despite this, little is known regarding the mitochondrial characteristics of chronically strength-trained individuals and any potential pathways regulating the strength-specific mitochondrial remodelling. Here, we investigated the mitochondrial structural characteristics in skeletal muscle of strength athletes and age-matched untrained controls. The mitochondrial pool in strength athletes was characterised by increased mitochondrial cristae density, decreased mitochondrial size, and increased surface-to-volume ratio, despite similar mitochondrial volume density. We also provide a fibre-type and compartment-specific assessment of mitochondria morphology in human skeletal muscle, which reveals across groups a compartment-specific influence on mitochondrial morphology that is largely independent of fibre type. Furthermore, we show that resistance exercise leads to signs of mild mitochondrial stress, without an increase in the number of damaged mitochondria. Using publicly available transcriptomic data we show that acute resistance exercise increases the expression of markers of mitochondrial biogenesis, fission and mitochondrial unfolded protein responses (UPRmt ). Further, we observed an enrichment of the UPRmt in the basal transcriptome of strength-trained individuals. Together, these findings show that strength athletes possess a unique mitochondrial remodelling, which minimises the space required for mitochondria. We propose that the concurrent activation of markers of mitochondrial biogenesis and mitochondrial remodelling pathways (fission and UPRmt ) with resistance exercise may be partially responsible for the observed mitochondrial phenotype of strength athletes. KEY POINTS: Untrained individuals and strength athletes possess comparable skeletal muscle mitochondrial volume density. In contrast, strength athletes' mitochondria are characterised by increased cristae density, decreased size and increased surface-to-volume ratio. Type I fibres have an increased number of mitochondrial profiles with minor differences in the mitochondrial morphological characteristics compared with type II fibres. The mitochondrial morphology is distinct across the subcellular compartments in both groups, with subsarcolemmal mitochondria being bigger in size when compared with intermyofibrillar. Acute resistance exercise leads to signs of mild morphological mitochondrial stress accompanied by increased gene expression of markers of mitochondrial biogenesis, fission and mitochondrial unfolded protein response (UPRmt ).

Mitochondrial Structure and Polarity in Dendrites and the Axon Initial Segment Are Regulated by Homeostatic Plasticity and Dysregulated in Fragile X Syndrome.

Mitochondrial dysfunction has long been overlooked in neurodevelopmental disorders, but recent studies have provided new links to genetic forms of autism, including Rett syndrome and fragile X syndrome (FXS). Mitochondria show plasticity in morphology and function in response to neuronal activity, and previous research has reported impairments in mitochondrial morphology and function in disease. We and others have previously reported abnormalities in distinct types of homeostatic plasticity in FXS. It remains unknown if or how activity deprivation triggering homeostatic plasticity affects mitochondria in axons and/or dendrites and whether impairments occur in neurodevelopmental disorders. Here, we test the hypothesis that mitochondria are structurally and functionally modified in a compartment-specific manner during homeostatic plasticity using a model of activity deprivation in cortical neurons from wild-type mice and that this plasticity-induced regulation is altered in Fmr1-knockout (KO) neurons. We uncovered dendrite-specific regulation of the mitochondrial surface area, whereas axon initial segment (AIS) mitochondria show changes in polarity; both responses are lost in the Fmr1 KO. Taken together, our results demonstrate impairments in mitochondrial plasticity in FXS, which has not previously been reported. These results suggest that mitochondrial dysregulation in FXS could contribute to abnormal neuronal plasticity, with broader implications to other neurodevelopmental disorders and therapeutic strategies.

Subcellular Specialization of 3D Mitochondrial Morphology and Mitochondria‐Lipid Droplet Interactivity is Associated with Proximity to Oxygen Supply in Muscle Cells

Advancements in imaging technology have considerably expanded our capacity to investigate cell biology, and our laboratory has recently employed high-resolution 3D microscopy to explore the subcellular structures and organelle-organelle interactions that support muscle metabolism. While this initial work has demonstrated that mitochondrial structure and mitochondria-organelle interactivity are regulated according to muscle cell type, it remains unclear how cellular metabolism is supported by the subcellular specialization of mitochondria. Certainly, several subcellular clusters of mitochondria have been identified within muscle cells, and these groups of mitochondria are often categorized according to their proximity to other cellular components (i.e. “intermyofibrillar” or “paravascular”). However, it is unknown how 3D mitochondrial morphology and organelle interactivity is altered to support the varying functional demands of different subcellular regions. Here, we combine 3D focused-ion beam scanning electron microscopy (FIB-SEM) with machine learning programs to quantify individual mitochondrial structure and organelle interactivity within large subcellular volumes (~8,000 µm3) (Fig. 1a-c). Using this high-throughput approach, we evaluate over 700 individual mitochondria per cell and leverage the statistical power of this large data set to identify 3D spatial relationships among multiple subcellular structures. We find that mitochondrial surface area to volume ratio (15.5±0.25 µm-1) is significantly correlated (R2= 0.39) to the minimal distance to capillary, but not distance to sarcolemma (R2= 0.02) (Fig. 1d). Further analysis revealed that mitochondria-organelle interactivity is also closely related to subcellular localization. Specifically, mitochondria within close proximity (<5 µm) to capillaries (paravascular) are ~3-fold more likely to make contact with lipid droplets compared to mitochondria located in other subcellular regions (Fig. 1e). Moreover, the paravascular mitochondria in contact with lipid droplets have 75% greater volume (1.4±0.07 µm3) compared to those not connected to lipid droplets (0.8±0.03 µm3). These preliminary analyses suggest that mitochondria in close proximity to the muscle oxygen supply, but not cell boundary, exhibit distinct 3D morphology and organelle interactivity patterns that may function to support metabolism and other cellular processes.

Postnatal development of the mitochondrial reticulum in the cardiac muscles of mice

Adult cardiac muscles are characterized by well-developed mitochondrial networks, which supports high energy demand in the heart. However, it is obscure how these structures are formed during postnatal development. Using focused ion beam scanning electron microscopy (FIB-SEM), we collected high-resolution image stacks of cardiac muscles of mice at postnatal (P) day 1, 7, 14, and 42, respectively (N=2-3 per group). Then, we conducted a semi-automated machine running to extract 3D mitochondrial formations and analyzed its morphological characteristics across postnatal days. After forming more stretched structures by P7, it was shown that cardiac muscles further develop spherical shaped-mitochondria and that they are overall arranged in the fiber-parallel direction. Although total mitochondrial volumes tend to increase across P1-P42, individual mitochondrial volumes were relatively smaller during early postnatal days (P1-P14) than in the matured muscles. Notably, we observed that individual mitochondrial surface area to volume ratios as well as aspect ratios are significantly downregulated during the entire developmental period by ~34% and ~31%, respectively. Furthermore, it was also shown that individual mitochondrial lengths are ~31% decreased across the postnatal days. In conclusion, these preliminary results provide dynamic transitions of individual mitochondrial structures during postnatal cardiac muscle development. Nevertheless, further analyses will be needed to study intermitochondrial interactions, as well as mitochondrial interactions with other subcellular components (e.g., lipid droplets), by which we can better understand functional aspects of mitochondria in developing cardiac muscles.

Grx2 Regulates Skeletal Muscle Mitochondrial Structure and Autophagy.

Glutathione is an important antioxidant that regulates cellular redox status and is disordered in many disease states. Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase that plays a pivotal role in redox control by catalyzing reversible protein deglutathionylation. As oxidized glutathione (GSSG) can stimulate mitochondrial fusion, we hypothesized that Grx2 may contribute to the maintenance of mitochondrial dynamics and ultrastructure. Here, we demonstrate that Grx2 deletion results in decreased GSH:GSSG, with a marked increase of GSSG in primary muscle cells isolated from C57BL/6 Grx2−/− mice. The altered glutathione redox was accompanied by increased mitochondrial length, consistent with a more fused mitochondrial reticulum. Electron microscopy of Grx2−/− skeletal muscle fibers revealed decreased mitochondrial surface area, profoundly disordered ultrastructure, and the appearance of multi-lamellar structures. Immunoblot analysis revealed that autophagic flux was augmented in Grx2−/− muscle as demonstrated by an increase in the ratio of LC3II/I expression. These molecular changes resulted in impaired complex I respiration and complex IV activity, a smaller diameter of tibialis anterior muscle, and decreased body weight in Grx2 deficient mice. Together, these are the first results to show that Grx2 regulates skeletal muscle mitochondrial structure, and autophagy.

A Diet High in Fat and Fructose Induces Early Hepatic Mitochondrial Aging.

To investigate the effect of high fructose diet on ultrastructure and content of hepatic mitochondria, we randomized 6-8 weeks old male C57Bl6/J mice to ad lib chow or high-fat-high-fructose (HF2) diet for 32 weeks. HF2-fed mice gained more weight, had higher plasma alanine aminotransferase, and fasting glucose levels and increased hepatic triglyceride content at all time points compared to chow-fed mice. HF2-fed mice had lower mitochondrial to nuclear DNA ratio compared to chow-fed mice. HF2-fed mice had lower average mitochondrial surface area and the number of mitochondria compared to chow-fed mice. HF2-fed mice had higher expression of the hepatic endoplasmic reticulum stress marker Chop, compared to chow-fed mice. A diet high in fat and fructose leads to enhanced hepatic mitochondrial aging, depletion, and dysfunction, which may be important determinants of nonalcoholic steatohepatitis pathogenesis.

The Functional Impact of Mitochondrial Structure Across Subcellular Scales.

Mitochondria are key determinants of cellular health. However, the functional role of mitochondria varies from cell to cell depending on the relative demands for energy distribution, metabolite biosynthesis, and/or signaling. In order to support the specific needs of different cell types, mitochondrial functional capacity can be optimized in part by modulating mitochondrial structure across several different spatial scales. Here we discuss the functional implications of altering mitochondrial structure with an emphasis on the physiological trade-offs associated with different mitochondrial configurations. Within a mitochondrion, increasing the amount of cristae in the inner membrane improves capacity for energy conversion and free radical-mediated signaling but may come at the expense of matrix space where enzymes critical for metabolite biosynthesis and signaling reside. Electrically isolating individual cristae could provide a protective mechanism to limit the spread of dysfunction within a mitochondrion but may also slow the response time to an increase in cellular energy demand. For individual mitochondria, those with relatively greater surface areas can facilitate interactions with the cytosol or other organelles but may be more costly to remove through mitophagy due to the need for larger phagophore membranes. At the network scale, a large, stable mitochondrial reticulum can provide a structural pathway for energy distribution and communication across long distances yet also enable rapid spreading of localized dysfunction. Highly dynamic mitochondrial networks allow for frequent content mixing and communication but require constant cellular remodeling to accommodate the movement of mitochondria. The formation of contact sites between mitochondria and several other organelles provides a mechanism for specialized communication and direct content transfer between organelles. However, increasing the number of contact sites between mitochondria and any given organelle reduces the mitochondrial surface area available for contact sites with other organelles as well as for metabolite exchange with cytosol. Though the precise mechanisms guiding the coordinated multi-scale mitochondrial configurations observed in different cell types have yet to be elucidated, it is clear that mitochondrial structure is tailored at every level to optimize mitochondrial function to meet specific cellular demands.

Mitochondrial architecture in cardiac myocytes depends on cell shape and matrix rigidity

Contraction of cardiac myocytes depends on energy generated by the mitochondria. During cardiac development and disease, the structure and function of the mitochondrial network in cardiac myocytes is known to remodel in concert with many other factors, including changes in nutrient availability, hemodynamic load, extracellular matrix (ECM) rigidity, cell shape, and maturation of other intracellular structures. However, the independent role of each of these factors on mitochondrial network architecture is poorly understood. In this study, we tested the hypothesis that cell aspect ratio (AR) and ECM rigidity regulate the architecture of the mitochondrial network in cardiac myocytes. To do this, we spin-coated glass coverslips with a soft, moderate, or stiff polymer. Next, we microcontact printed cell-sized rectangles of fibronectin with AR matching cardiac myocytes at various developmental or disease states onto the polymer surface. We then cultured neonatal rat ventricular myocytes on the patterned surfaces and used confocal microscopy and image processing techniques to quantify sarcomeric α-actinin volume, nucleus volume, and mitochondrial volume, surface area, and size distribution. On some substrates, α-actinin volume increased with cell AR but was not affected by ECM rigidity. Nucleus volume was mostly uniform across all conditions. In contrast, mitochondrial volume increased with cell AR on all substrates. Furthermore, mitochondrial surface area to volume ratio decreased as AR increased on all substrates. Large mitochondria were also more prevalent in cardiac myocytes with higher AR. For select AR, mitochondria were also smaller as ECM rigidity increased. Collectively, these results suggest that mitochondrial architecture in cardiac myocytes is strongly influenced by cell shape and moderately influenced by ECM rigidity. These data have important implications for understanding the factors that impact metabolic performance during heart development and disease.

PS421 huntingtin modulates mitochondrial phenotypes and confers neuroprotection in an HD hiPSC model

Huntington disease (HD) is a hereditary neurodegenerative disorder caused by mutant huntingtin (mHTT). Phosphorylation at serine-421 (pS421) of mHTT has been shown to be neuroprotective in cellular and rodent models. However, the genetic context of these models differs from that of HD patients. Here we employed human pluripotent stem cells (hiPSCs), which express endogenous full-length mHTT. Using genome editing, we generated isogenic hiPSC lines in which the S421 site in mHTT has been mutated into a phospho-mimetic aspartic acid (S421D) or phospho-resistant alanine (S421A). We observed that S421D, rather than S421A, confers neuroprotection in hiPSC-derived neural cells. Although we observed no effect of S421D on mHTT clearance or axonal transport, two aspects previously reported to be impacted by phosphorylation of mHTT at S421, our analysis revealed modulation of several aspects of mitochondrial form and function. These include mitochondrial surface area, volume, and counts, as well as improved mitochondrial membrane potential and oxidative phosphorylation. Our study validates the protective role of pS421 on mHTT and highlights a facet of the relationship between mHTT and mitochondrial changes in the context of human physiology with potential relevance to the pathogenesis of HD.

3D mitochondrial network formation during postnatal skeletal muscle development of mice

Mitochondrial connectivity plays an important role for muscle energy distribution. However, it is obscure how their structural formation is developed during postnatal muscle growth. Here, using a high‐resolution electron microscopy (i.e., FIB‐SEM), we have sought to examine three dimensional (3D) mitochondrial network formations in oxidative (soleus) and glycolytic (gastrocnemius) muscles of C57BL/6N mice at postnatal (P) 1, 7, 14, 21, and 42 day, respectively. After semi‐automated machine learning process, 3D mitochondrial reticulum formations were rendered and quantitatively analyzed by ImageJ software. Both types of skeletal muscles showed similarly fiber parallel mitochondrial network formations during early days (P1–P7). After P14, oxidative muscles developed grid‐like mitochondrial reticulum, while glycolytic muscle mitochondria were found as perpendicular formations along the Z‐lines. Unlikely to glycolytic muscles where individual mitochondrial volumes were similarly maintained across the development, oxidative muscles significantly increased those volumes by P14 and then largely decreased to the level comparable to glycolytic muscles. After P14, oxidative muscles maintained notably higher total mitochondrial volumes as compared to glycolytic muscles. Furthermore, we observed that at P14, glycolytic muscles showed remarkably higher ratio of mitochondrial surface area to volume, as well as sphericity, than oxidative muscles. Another interesting point was that during early muscle growth (P1–P7), mitochondrial elongation, aspect ratio, and length were all shown to be higher in glycolytic muscles than in oxidative muscles. In conclusion, this preliminary study provides how 3D mitochondrial networks are changed over the course of postnatal muscle development, and also suggests that P14 may be a critical time point for the mitochondrial transformation. However, further studies are needed to better understand 3D muscle mitochondrial development along with contractile components and other subcellular elements such as lipid droplets and sarcoplasmic reticulum.Support or Funding InformationThis work is supported by the Division of Intramural Research of the National Heart, Lung and Blood Institute and the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Overexpression of Optic Atrophy Type 1 Protects Retinal Ganglion Cells and Upregulates Parkin Expression in Experimental Glaucoma.

Glaucoma is a neurodegenerative disease that features progressive loss of retinal ganglion cells (RGCs). Increasing evidences have revealed that impaired mitochondrial dynamics occurs early in neurodegenerative diseases. Optic Atrophy Type 1 (OPA1), a mitochondrial fusion protein, has recently been suggested to be a mitophagic factor. Our previous studies found that glaucomatous retinal damage may be ameliorated by an increase in mitochondrial OPA1. In this study, we explored the mechanism involved in OPA1 mediated neuroprotection and its relationship with parkin dependent mitophagy in experimental glaucoma models. Our data showed that overexpression of OPA1 by viral vectors protected against RGC loss, attenuated Bax expression, and improved mitochondrial health and mitochondrial surface area. Parkin expression and the number of mitophagosomes were upregulated in OPA1 overexpressed RGCs under glutamate excitotoxicity. While knockdown of OPA1 by siRNA decreased protein expression of parkin in RGCs under glutamate excitotoxicity. Two weeks after intraocular pressure (IOP) elevation, the LC3-II/I ratio and the LAMP1 expression were increased in OPA1 overexpressed optic nerve. These findings suggest that OPA1 overexpression may protect RGCs by ways of enhancing mitochondria fusion and parkin mediated mitophagy. Interventions to promote mitochondrial fusion and mitophagy may provide a useful strategy to battle against glaucomatous RGC loss.

Abstract 375: Origins of fractional control in regulated cell death

Abstract Fractional killing of a population of cancer cells is often linked to cell-to-cell variability due to stochastic gene expression and fluctuations in protein levels. We found that mitochondria abundance is a major source of cell-to-cell variability that determines the fraction of cells that live or die in response to TNF-related apoptosis-inducing ligand (TRAIL). We show that the IC50 of TRAIL of clonogenic cancer cell populations correlates with mitochondria abundance, and cell-to-cell variability in mitochondria abundance controls up to 40% of the response to TRAIL. Our coarse-grained quantitative model of apoptosis reveals that increasing the mitochondrial surface area decreases the density of proapoptotic Bax pores, which in turn decreases the sensitivity of cells to TRAIL. We demonstrate that the clinically relevant small-molecule inhibitor of antiapoptotic Bcl-2 proteins ABT-263 reduces the IC50 of TRAIL by reducing the contribution of mitochondria to variability in apoptosis. Citation Format: Luis C. Santos, Robert Vogel, Jerry E. Chipuk, Marc Birtwistle, Gustavo Stolovitzky, Pablo Meyer. Origins of fractional control in regulated cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 375.

Oleanolic Acid, a Novel Endothelin A Receptor Antagonist, Alleviated High Glucose-Induced Cardiomyocytes Injury.

Endothelin-1 (ET-1) and its receptor endothelin A receptor (ET[Formula: see text] have been shown to be upregulated in a high glucose environment, which increase the incidence of diabetes-related heart failure. Our previous study demonstrated that oleanolic acid (OA), a natural compound found in Chinese herbs had ET-1 antagonistic effects. We aimed to verify whether OA could ameliorate diabetes mellitus (DM)-induced injury in cardiomyocytes by reducing the antagonistic effects of the ET-1 pathway. For the induction of high glucose-related injury in cardiomyocytes, neonatal rat ventricular cardiomyocytes (NRVMs) were subjected to culture medium containing 25[Formula: see text]mM of glucose. Natriuretic peptide B (BNP), mitochondrial membrane potential (MMP) and cell surface area were measured to evaluate the severity of NRVMs injury. mRNA expression of ET-1 and ETA was determined using quantitative PCR. Moreover, a Ca[Formula: see text] influx assay was used to evaluate potential ETA antagonistic effects. Molecular docking of OA and ETA was performed using the Sulflex-Dock program. Human induced pluripotent stem cell (iPS-C)-derived cardiomyocytes and real time cell analysis system (RTCA) were used to verify the effect of OA on the ET-1 pathway. High glucose levels increased the expression of BNP at both mRNA and protein levels in cardiomyocytes. Moreover, cell surface area and MMP were also elevated in a high glucose environment. High glucose-induced injury in NRVMs was not reversible by hypoglycemic therapy. In addition, ETA was upregulated by high glucose treatment and levels could not be reduced by hypoglycemic treatment. The Ca[Formula: see text] influx assay on ETA/HEK293 cells showed that OA had a partial ETA antagonistic effect. Molecular docking approaches showed that OA was docked into the active site of ETA. Furthermore, functionality tests based on iPS-C and RTCA demonstrated that treatment with OA could reverse ET-1-induced alternation of beating rates and amplitude. Thus, OA could reverse high glucose-induced BNP upregulation, and increased both the cell area and MMP in NRVMs. High glucose-induced irreversible ETA upregulation is a major reason of continuous diabetes-related injury in cardiomyocytes. Treatment with OA had a protective effect on high glucose-induced injury in cardiomyocytes through a partial ETA antagonistic role.

On Some Gastrointestinal Effects of Khat ( Catha edulis )

Background: The possible gastrointestinal effects of khat (Catha edulis) chewing still under debate. Some authors are on the opinion that khat has a gastric and/or duodenal ul-cerogenic effect, while others denies this effect. Considering the absolutely different standpoints in this case, we have decided to investigate this contradiction in different experimental ulcer models of rat. Materials and Methods: It is known that the main effective (amphetamine-like) constituent of the khat is cathinone, we have investigated the effect of 500 and 1000 ug/kg of its dose in stress (restrain) and drug (indomethacine) induced gastric, as well, as cyste-amine induced duodenal ulcer-models. It seems that in indomethacine and in stress induced models cathinone showed no ulcerogenic effect, moreover in the indomethacine induced model it showed a significant anti-ulcerogenic effect. Moreover, scanning electron microscopic investigations verified that the acute cathinone treatment has no detectable effect on gastric mucosa. In the cysteamine-induced duodenal ulcer-model cathinon showed neither ulcerogenic, nor antiulcerogenic effect. Discussion and Conclusion: Moreover, our scanning electron microscopic investigations showed that basically no structural changes can be seen in the treated animals’ liver; the only visible change is that the mitochondrial surface area of the treated liver is enlarged. According to our opinion our experimental results presented, gave evidence that khat, at least its cathinone content, have no gastrointestinal (ulcerogenic) effect.

Contributions of photosynthetic and non-photosynthetic cell types to leaf respiration in Vicia faba L. and their responses to growth temperature.

In intact leaves, mitochondrial populations are highly heterogeneous among contrasting cell types; how such contrasting populations respond to sustained changes in the environment remains, however, unclear. Here, we examined respiratory rates, mitochondrial protein composition and response to growth temperature in photosynthetic (mesophyll) and non-photosynthetic (epidermal) cells from fully expanded leaves of warm-developed (WD) and cold-developed (CD) broad bean (Vicia faba L.). Rates of respiration were significantly higher in mesophyll cell protoplasts (MCPs) than epidermal cell protoplasts (ECPs), with both protoplast types exhibiting capacity for cytochrome and alternative oxidase activity. Compared with ECPs, MCPs contained greater relative quantities of porin, suggesting higher mitochondrial surface area in mesophyll cells. Nevertheless, the relative quantities of respiratory proteins (normalized to porin) were similar in MCPs and ECPs, suggesting that ECPs have lower numbers of mitochondria yet similar protein complement to MCP mitochondria (albeit with lower abundance serine hydroxymethyltransferase). Several mitochondrial proteins (both non-photorespiratory and photorespiratory) exhibited an increased abundance in response to cold in both protoplast types. Based on estimates of individual protoplast respiration rates, combined with leaf cell abundance data, epidermal cells make a small but significant (2%) contribution to overall leaf respiration which increases twofold in the cold. Taken together, our data highlight the heterogeneous nature of mitochondrial populations in leaves, both among contrasting cell types and in how those populations respond to growth temperature.

Mitochondrial morphology differences and mitophagy deficit in murine glaucomatous optic nerve.

Decreased ATP correlates with intraocular pressure exposure in the optic nerves of mice with glaucoma. To understand what underlies this energy deficit, we examined mitochondria in the myelinated optic nerve axons of the DBA/2J mouse, a model of glaucoma secondary to iris pigment disease, and the DBA/2(wt-gpnmb) control strain. Mitochondrial length, width, surface area, and health status were measured in 30 electron microscopic fields within the myelinated portion of optic nerves from DBA/2J and DBA/2(wt-gpnmb) mice at 3, 6, and 10 months of age. Protein was isolated from optic nerve for analysis of PINK1, Parkin, LC3-I and -II, and lysosome-associated membrane protein 1 (LAMP1) by Western blot. The number of mitochondria in DBA/2J optic nerve was increased, and they had significantly smaller surface area. Mitochondria in DBA/2J were closer to the axolemma, more spatially isolated, and their cristae were more disrupted at every age group as compared to DBA/2(wt-gpnmb). Autophagosomes were significantly increased in DBA/2J optic nerve at all ages. Protein analysis showed higher LC3-II to LC3-I ratio in aged DBA/2J optic nerve than in DBA/2(wt-gpnmb). PINK1 and Parkin levels were not statistically different across age groups. LAMP1 was significantly decreased in the aged DBA/2J optic nerve. Decreased surface area, combined with reduced oxidative capacity in mitochondria from the aged DBA/2J axon, indicate that mitochondrial pathology may contribute to the energy deficit in glaucomatous optic nerve. Though autophagosomes were increased in DBA/2J optic nerve, the increased mitochondria and decreased LAMP1 suggest deteriorating mitochondria are not being efficiently recycled by mitophagy.

Diabetes reduces β-cell mitochondria and induces distinct morphological abnormalities, which are reproducible by high glucose in vitro with attendant dysfunction

We investigated the impact of a diabetic state with hyperglycemia on morphometry of β cell mitochondria and modifying influence of a K+-ATP channel opener and we related in vivo findings with glucose effects in vitro. For in vivo experiments islets from syngeneic rats were transplanted under the kidney capsule to neonatally streptozotocin-diabetic or non-diabetic recipients. Diabetic recipients received vehicle, or tifenazoxide (NN414), intragastrically for 9 weeks. Non-diabetic rats received vehicle. Transplants were excised 7 d after cessation of treatment (wash-out) and prepared for electron microscopy. Morphological parameters were measured from approx. 25,000 mitochondria. Rat islets were cultured in vitro for 2–3 weeks at 27 or 11 (control) mmol/l glucose. Transplants to diabetic rats displayed decreased numbers of mitochondria (-31%, p < 0.05), increased mitochondrial volume and increased mitochondrial outer surface area, p < 0.001. Diabetes increased variability in mitochondrial size with frequent appearance of mega-mitochondria. Tifenazoxide partly normalized diabetes-induced effects, and mega-mitochondria disappeared. Long-term culture of islets at 27 mmol/l glucose reproduced the in vivo morphological abnormalities. High-glucose culture was also associated with reduced ATP and ADP contents, reduced oxygen consumption, reduced signaling by MitoTracker Red and reduction of mitochondrial proteins (complexes I–IV), OPA 1 and glucose-induced insulin release. We conclude that (1) a long-term diabetic state leads to a reduced number of mitochondria and to distinct morphological abnormalities which are replicated by high glucose in vitro; (2) the morphological abnormalities are coupled to dysfunction; (3) K+-ATP channel openers may have potential to partly reverse glucose-induced effects.

Comparative study of 3D morphology and functions on genetically engineered mouse melanoma cells

Quantification of 3D morphology and measurement of cellular functions were performed on the mouse melanoma cell lines of B16F10 to investigate the intriguing problem of structure-function relations in the genetically engineered cells with GPR4 overexpression. Results of 3D analysis of cells in suspension and phase contrast imaging of adherent cells yield consistent evidence that stimulation of the proton-sensing GPR4 receptor in these cells may modify significantly their morphology with diminishing ability to produce membrane protrusions and to migrate. Examination of the 3D parameters of mitochondria provide further insights on the measured variation of the maximal capacity of oxygen consumption rate among the genetically modified cells, indicating that the proton-sensing receptor may regulate cancer cell metabolism with increased mitochondrial surface area. Our study demonstrates clearly the significant benefits of quantitative 3D morphological study in illuminating cellular functions and development of novel morphology based cell assay methods.

Should Willy Wonka have been a sports nutritionist?

Should Willy Wonka have been a sports nutritionist?

Steatosis of indeterminate cause in a pediatric group: is it a primary mitochondrial hepatopathy?

In children, hepatic steatosis may be related to inborn errors of metabolism (IEMs) or to non-alcoholic fatty liver disease (NAFLD). The aim of this study was to assess and characterize steatosis of indeterminate cause through morphological and morphometric analysis of liver tissue. Cross-sectional study at the Departments of Pathology of Faculdade de Ciências Médicas, Universidade Estadual de Campinas (FCM-Unicamp) and Faculdade de Medicina de Botucatu, Universidade Estadual Paulista (FMB-Unesp). Eighteen consecutive liver biopsies obtained from 16 patients of ages ranging from 3 months to 12 years and nine months that were inserted in a database in the study period were analyzed using optical microscopy and transmission electron microscopy. Through electron microscopy, the mitochondrial density and mean mitochondrial surface area were determined in hepatocytes. Ten patients ranging in age from 1 to 14 years were used as a control group. "Pure" steatosis was detected, unaccompanied by fibrosis or any other histological alteration. Microvesicular steatosis predominated, with a significant increase in mean mitochondrial surface area. Microvesicular steatosis may be related to primary mitochondrial hepatopathy, especially due to reduction of β-oxidation or partial stagnation of oxidative phosphorylation. For these reasons, this form of steatosis (which should not be called "pure") is likely to represent an initial stage in the broad spectrum of NAFLD. We have drawn attention to cases of steatosis in the pediatric group, in which the microvesicular form predominates, since this may be associated with mitochondrial disorders.