Mitochondrial ultrastructure in cardiomyocytes from 3- and 24-month-old Wistar and OXYS rats was investigated using a new approach designed for morphometric analysis. The data fully confirm the electron microscopy data: the area of the inner mitochondrial membrane per unit volume of mitochondria was significantly decreased with age, as found on heart muscle section. In 3-month-old Wistar rats from the control group, this parameter was 41.3 ± 1.52 µm(2)/µm(3), whereas in OXYS rats it was decreased to 30.57 ± 1.74 µm(2)/µm(3). With age, an area of the inner mitochondrial membrane per unit volume of mitochondria declined in both rat strains: Wistar - from 41.3 ± 1.52 to 21.47 ± 1.22 µm(2)/µm(3), OXYS - from 30.57 ± 1.74 to 16.3 ± 0.89 µm(2)/µm(3). A new method that we designed and used for morphometric analysis notably simplifies the process of morphometric measurements and opens up good opportunities for its further optimization using image recognition technology.

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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 µm 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 µm. 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.

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.

158Starting from 1961, two models for transforma tion of the energy of oxidation reactions in mito chondria have been discussed in the literature. The model of Mitchell [1] implies that the multienzyme phosphorylation system can work in a dissociated form, while the other model (by Williams) considers a supercomplex [2].We have earlier demonstrated that the phosphory lation system is able to function in both modes, corre sponding to two structural states of mitochondria [3]. The operation modes of the phosphorylating system are provided by the system of mitochondrial volume regulation [4,5].In hypotonic media under conditions of low amplitude mitochondrial swelling, the Williams model considers protons (unstably bound to the membrane supercomplex as Brønsted acids) as a substrate for ATP synthase.In 1976, we demonstrated the possibility to use a Brønsted acid as a substrate for ATP synthase using a model system. In these experiments, ATP synthase was sorbed at the octane-water interface. In this sys tem, ATP synthesis was observed in the presence of ADP and phosphate, and the gradient of a Brønsted acid (pentachlorophenol) at the octane-water inter face was used as a source of energy; the gradient was created by adding pentachlorophenol to the octane phase [6].On the bilayer membrane, we have found the cata lysts that selectively accelerate dissociation of Brøn sted acids [7]. This allowed us to find [8] that a non equilibrium fraction of Brønsted acids with excess free energy was formed on the surface of mitoplasts (mito chondria without the outer membrane), provided that H + pumps function.The goal of this work was to prove the formation of Brønsted acids during ATP synthesis on the mito chondrial membranes.A tightly joining of the cristae (Figs. 1 and 2) and, correspondingly, the joining of the outer and inner membranes should be expected. In this process, a two membrane structure of mitoplasts is formed of the outer and inner membranes. Since the outer mem brane is well permeable ("transparent") for hydrogen ions in course of proton pumping, this structure should function as an integral whole interacting with hydrogen ions, possessing excess free energy (accord ing to the scheme in Fig. 1b).We have earlier demonstrated that the measure ment of surface ζ potential (using a Zetasizer Nano ZS device) of mitoplasts can be efficiently used for detecting formation of nonequilibrium Brønsted acids in functional states [8].To selectively accelerate dissociation of mem brane bound Brønsted acids, possessing excess free energy, we increased the concentration of catalyst (HEPES) [7].The measurements were performed in the media with a tonicity of 350 and 120 mOsm. In this experi ment, we compared the measured ζ potential on the surface of functional mitochondria during ATP syn thesis under isotonic and hypotonic conditions. The experiments conducted under hypotonic conditions have demonstrated (Table 1) that the changes in ζ potential on the mitochondrial surface regularly cor relate with the changes in ζ potential on t...

We studied age-related ultrastructural reorganization in acinar cells and intercalary ducts of the lacrimal gland acini in 3-, 15-, and 24-month-old Wistar rats. Ultrastructural changes in the lacrimal gland progressed with age and led to dramatic ultrastructural reconstruction of the lacrimal gland at the age of 24 months. These changes mainly included complete destruction of acinar cells and increase in the number of enlarged branched ducts that filled the greater part of gland volume; these dusts were lined with epithelial cells with altered ultrastructure. Acinar cells in the acini communicate via special connecting intermembrane complexes formed by desmosomes and mitochondria adjacent to them in each contacting cell. It is assumed that association of mitochondria with desmosomes found in the acini is a special functional complex indicating that every single acinus is a functional formation. This assumption is indirectly confirmed by the fact that the destruction never occurred in a single cell, but always involved all cells constituting the acini. The revealed ultrastructural changes reflect age-related deterioration of the secretory function of the lacrimal gland.

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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.