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.

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.

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.

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.

Autophagy is a conserved lysosome-dependent process essential for cellular homeostasis, and pharmacological activation of autophagy has emerged as a promising therapeutic strategy for age-related, neurodegenerative, metabolic, and malignant diseases. Despite the growing interest in natural autophagy modulators, lichens remain largely unexplored as a source of autophagy-inducing compounds. Here, we tested ethanolic extracts from four commercially available lichens using LC3-II accumulation as a primary readout. Usnea sp. was identified as the most active species. Activity-guided fractionation followed by HRMS and NMR analyses identified salazinic acid as the active metabolite. Salazinic acid increased LC3-II accumulation, enhanced autophagic flux in the presence of bafilomycin A1, and promoted autophagosome formation as confirmed by transmission electron microscopy. The compound exhibited low cytotoxicity in human cell lines at concentrations exceeding those required for autophagy induction. Mechanistically, salazinic acid activated the phosphorylation of AMPK, ULK1, and AKT; however, it retained its ability to stimulate autophagic flux in AMPK- and ULK1-deficient cells, indicating that its activity is not strictly dependent on the canonical AMPK–ULK1 signaling pathway. Collectively, our findings identify salazinic acid as a previously unrecognized inducer of productive autophagic flux and demonstrate that lichen secondary metabolites represent a promising and largely untapped source of novel autophagy-modulating compounds.

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.