Lora E. Bakeeva

Cardiac pacemaker and valve cells operate within a heterogeneous interstitial environment, but the identities and observable behaviors of many constituent cells remain incompletely defined. We integrated complementary imaging observations from a single experimental platform into an exploratory analysis of cultured cells isolated from the adult mouse sinoatrial node (SAN) and atrioventricular valves of S100B-EGFP reporter mice. Cellular morphology, endogenous NAD(P)H fluorescence, tetramethylrhodamine methyl ester (TMRM) fluorescence, correlative light and electron microscopy (CLEM), time-lapse imaging, transmission electron microscopy (TEM), and 5-ethynyl-2′-deoxyuridine (EdU) labeling were used to describe selected reporter-positive and reporter-negative cells. S100B-promoter-associated EGFP+ cells displayed heterogeneous forms, including cells with small bodies and long processes. In representative fields, EGFP+ cells showed greater NAD(P)H autofluorescence intensity and lower TMRM signal than adjacent EGFP− cells. CLEM related selected fluorescence phenotypes to ultrastructure in the same cells and documented mitochondrial, vesicular, nuclear, and membrane-associated profiles. One live sequence captured movement of a TMRM-positive, mitochondrion-like structure through a thin intercellular bridge toward an EGFP− cell, followed by bridge disassembly. Additional images showed mitochondrial and vesicular profiles near cell surfaces or in extracellularly situated regions, elongated NAD(P)H-bright structures, reporter-positive cytoplasmic fragments, nuclear size heterogeneity, a narrow connection between nuclear profiles, and unequal EdU labeling among nuclear fragments. These findings are descriptive and hypothesis-generating. They do not establish definitive cell identity, phenotype prevalence, active or selective secretion, tunneling-nanotube identity, mitochondrial metabolic competence, regulated nuclear remodeling, recipient-cell uptake, or physiological consequence. The study provides an integrated morphological framework and a set of candidate events for future quantitative investigation of S100B-associated cardiac interstitial-cell biology.

In this study, in a culture of renal epithelial cells, we identified those expressing nestin, a cytoskeletal protein associated with stem/progenitor/activated/proliferating cell states. A mouse expressing GFP under the nestin promoter was used, followed by cell isolation and culture. It is hypothesized that this can be used to assess the stem/progenitor/activated/proliferating cell level in a mixed kidney cell culture. Both nestin-positive and nestin-negative cells were demonstrated to be present in the culture. After visualization, cells were attached to a glass slide with a grid, fixed, and prepared for electron microscopy analysis, with each cell visually identified by light microscopy being analyzed. Electron microscopy revealed tight interactions between nestin-positive and nestin-negative cells. Significant differences in the ultrastructure of nestin-positive and nestin-negative cells were observed. Nestin-positive cells were distinguished by a high ribosome content, indicating high protein-synthesizing activity. In the nestin–GFP-high (sorted) population examined by electron microscopy, vesicle-containing protrusions were frequently observed. These cells could contain multiple nuclei of varying sizes and had a high content of lysosomes. No significant differences in mitochondrial ultrastructure were observed in nestin-positive and -negative cells, although functional characteristics evaluated by the membrane potential probe differed.

The reason for the exceptional longevity of the naked mole rat (Heterocephalus glaber) remains a mystery to researchers. We assumed that evolutionarily, H. glaber acquired the ability to quickly stabilize the functioning of mitochondria and endoplasmic reticulum (ER) to adjust metabolism to external challenges. To test this, a comparison of the hepatic mitochondria and ER of H. glaber and C57BL/6 mice was done. Electron microscopy showed that 2-months-old mice have more developed rough ER (RER) than smooth ER (SER), occupying ~17 and 2.5% of the hepatocytic area correspondingly, and these values do not change with age. On the other hand, in 1-week-old H. glaber, RER occupies only 13% constantly decreasing with age, while SER occupies 35% in a 1-week-old animal, constantly rising with age. The different localization of mitochondria in H. glaber and mouse hepatocytes was confirmed by confocal and electron microscopy: while in H. glaber, mitochondria were mainly clustered around the nucleus and on the periphery of the cell, in mouse hepatocytes they were evenly distributed throughout the cell. We suggest that the noted structural and spatial features of ER and mitochondria in H. glaber reflect adaptive rearrangements aimed at greater tolerance of the cellular system to challenges, primarily hypoxia and endogenous and exogenous toxins. Different mechanisms of adaptive changes including an activated hepatic detoxification system as a hormetic response, are discussed considering the specific metabolic features of the naked mole rat.

A comparative electron microscopic and morphometric analysis of age-related transformations in the ultrastructure of the skeletal muscle mitochondrial apparatus was carried out in animal species with different aging programs: short-lived classical objects, such as C57BL/6 mice and Wistar rats, prematurely aging OXYS rats, and the longest-living rodents, naked mole-rats (Heterocephalus glaber), characterized by delayed aging. In C57BL/6 mice, age-related reorganization of the skeletal muscle mitochondrial apparatus corresponds to that reported previously for Wistar rats: the mitochondrial reticulum forms by the age of 2.5–3 months; by the age of 30 months, it undergoes a drastic reduction, due to which the number of mitochondrial cross-sections in muscle fibers decreases almost twofold, from 0.45 ± 0.074 to 0.23 ± 0.017 profiles per µm². In C57BL/6 mice, no destructive changes in the mitochondrial ultrastructure were observed, in contrast to OXYS rats, in which age-related changes in the chondriome affect both the overall structure and internal ultrastructure of the muscle fiber mitochondrial apparatus. At the same time, in naked mole-rats, which are comparable with mice in their size, the number and size of mitochondria in skeletal muscles increase significantly by the age of five years, although no mitochondrial reticulum forms. It is hypothesized that a special organization of the mitochondrial apparatus in naked mole-rat skeletal muscles provide a proper level of redox processes in muscles, thus preventing a decline in their physical efficiency and the development of sarcopenia, whereas in C57BL/6 mice, Wistar and OXYS rats, age-related abnormalities in the structural organization of skeletal muscle mitochondrial apparatus may be one of the major causes for the development of age-related pathologies, including sarcopenia.

The significant destructive changes in ultrastructure of hepatocytes from laboratory mice kept in different vivariums in Moscow and fed with dry laboratory animal diets acquired from different domestic manufacturers that were not standardized for initial products were demonstrated using electron microscopy. Furthermore, disruption in the ultrastructure of liver parenchymal cells occurred regardless of the animal status (SPF or conventional), conditions of various vivariums, as well as the feed manufacturer. At the same time, studies on ultrastructure of liver hepatocytes from mice kept in the Charles River Laboratory facilities in Germany and fed with the Altromin Spezialfutter laboratory animal diet (GmbH & Co., Germany) that was produced using quality control of ingredients did not reveal destructive changes in the internal ultrastructure of hepatocytes. However, if these mice were later fed with the food produced in local manufactures, changes in the structure of liver cells developed after 2 months. Thus, feeding with dry diet from the domestic producers of an unspecified composition causes significant changes in the ultrastructure of hepatocytes in control animals, reflecting the development of some pathological processes in the body.

Internal ultrastructure of the muscle tissue mitochondria of horsehair worm Gordionus alpestris (Nematomorpha) was studied using morphometry. Surface area of the inner mitochondrial membrane per unit of the mitochondrial volume, or surface density of the inner mitochondrial membrane, was measured as a main morphometric parameter. The surface density of the inner mitochondrial membrane of the G. alpestris muscle tissue was compared to the respective parameter of the skeletal and cardiac muscle mitochondria. The surface density of the inner mitochondrial membrane of the worm was close to the surface density values of the cardimyocytes of 3-month-old mice and Wistar rats and was slightly higher than the surface density of mitochondria from the skeletal muscle of 3-month-old mice. The functional significance of the well-developed system of mitochondrial membranes of extended mitochondria of the horsehair worm is discussed as a structure necessary to ensure effective functioning of the circomyarian conntractile apparatus in the muscle tissue of the horsehair worm.

Electron microscopic study of cardiomyocytes taken from healthy Wistar and OXYS rats and naked mole rats (Heterocephalus glaber) revealed mitochondria in nuclei that lacked part of the nuclear envelope. The direct interaction of mitochondria with nucleoplasm is shown. The statistical analysis of the occurrence of mitochondria in cardiomyocyte nuclei showed that the percentage of nuclei with mitochondria was roughly around 1%, and did not show age and species dependency. Confocal microscopy of normal rat cardiac myocytes revealed a branched mitochondrial network in the vicinity of nuclei with an organization different than that of interfibrillar mitochondria. This mitochondrial network was energetically functional because it carried the membrane potential that responded by oscillatory mode after photodynamic challenge. We suggest that the presence of functional mitochondria in the nucleus is not only a consequence of certain pathologies but rather represents a normal biological phenomenon involved in mitochondrial/nuclear interactions.

In this study, the ultrastructure of mitochondria in cardiomyocytes of naked mole rats (Heterocephalus glaber) aged from 6 months to 11 years was examined. Mitochondria in cardiomyocytes of naked mole rats have a specific ultrastructure that is different from those in cardiomyocytes of other mammalian species studied to date. In contrast to mitochondria of other mammalian cardiomyocytes, where the internal space is completely filled by tightly packed parallel rows of cristae, mitochondria in cardiomyocytes of naked mole rats have a chaotic pattern of cristae organization with wave-like contours. Gradual formation of mitochondrial ultrastructure occurs in naked mole rats for many years. Two mitochondrial populations are developed to the age of 5 years. In addition to the main population, there are some large organelles which exceed normal sizes by two to three times. Most cristae in these mitochondria are assembled into small groups, which form the curved and ring-like structures. The appearance of some specific structural changes (i.e. bundles of parallel cristae) is observed in the mitochondrial population of naked mole rat after 11 years of age. However, these bundles are very rare and of sporadic nature. Morphometric analysis has shown that the superficial density of the inner mitochondrial membrane is similar in all examined age groups of naked mole rats: 21.1 at 6 months; 23.21 at 3 years; 23.55 at 5 years; and 20.8 at 11 years. This level is almost two times lower than in other animals studied (mice and rats). The data demonstrate that pathological changes in mitochondrial apparatus are not present in naked mole rats at least until the age of 11 years. The mitochondrial apparatus corresponds to the phenotype in young animals, thus being another neotenic feature in naked mole rats.

Morphometric analysis of mitochondria in skeletal muscles and heart of 6- and 60-month-old naked mole rats (Heterocephalus glaber) revealed a significant age-dependent increase in the total area of mitochondrial cross-sections in studied muscle fibers. For 6- and 60-month-old animals, these values were 4.8 ± 0.4 and 12.7 ± 1.8%, respectively. This effect is mainly based on an increase in the number of mitochondria. In 6-month-old naked mole rats, there were 0.23 ± 0.02 mitochondrial cross-sections per µm2 of muscle fiber, while in 60-month-old animals this value was 0.47 ± 0.03. The average area of a single mitochondrial cross-section also increased with age in skeletal muscles - from 0.21 ± 0.01 to 0.29 ± 0.03 µm2. Thus, naked mole rats show a drastic enlargement of the mitochondrial apparatus in skeletal muscles with age due to an increase in the number of mitochondria and their size. They possess a neotenic type of chondriome accompanied by specific features of mitochondrial functioning in the state of oxidative phosphorylation and a significant decrease in the level of matrix adenine nucleotides.

For many years, investigators have attempted to identify unique ultrastructural conditions of mitochondria related to aging. However, this did not result in definitive results. At present, this issue has again become of topical interest due to development of the mitochondrial theory of aging and of engineering of a novel antioxidant class known as mitochondria-targeted antioxidants. The review briefly discusses experimental results that, from our perspective, allow the most objective understanding regarding age-related changes in mitochondrial ultrastructure.