性成熟早产大鼠左心室心肌细胞的电子显微镜研究

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Background

Prematurity is linked to the early development of cardiovascular diseases [1]. Recent research has aimed to establish the structural basis for functional cardiac alterations in adolescents and adults who were born prematurely. Premature infants (born between 22 and 37 weeks of gestation) exhibit more spherical hearts in adolescence and adulthood [2] than their full-term counterparts. Additionally, their myocardium exhibits more pronounced interstitial fibrosis [3, 4]. However, clinical investigations alone are insufficient to establish an understanding of the histological and ultrastructural features of the myocardium of prematurely born individuals in adolescence and adulthood. Hence, there is a need for additional experimental research focused on this issue.

This study aimed to identify the ultrastructural features of left ventricular cardiomyocytes in prematurely born mature rats.

Materials and Methods

Study design

The study was prospective, experimental, single-center, randomized, controlled, and non-blinded.

Eligibility criteria

The investigation was conducted on male Wistar rats that were full-term and 24-hour premature. Based on the duration of intrauterine development, we classified the animals into two groups: full-term and premature, with Wistar rats having a total gestation period of 22 days.

Settings

The study was conducted at the Siberian State Medical University of the Ministry of Health of the Russian Federation. Standard vivarium conditions were implemented to effectively maintain the animals.

Duration of the study

The study was conducted on day 180 of postnatal ontogenesis in Wistar rats to ascertain the long-term effects of prematurity on the structural elements of cardiomyocytes.

Description of medical intervention

The offspring were obtained from mature Wistar male (2 months old, 200 g) and female (3 months old, 200 g) rats. The following morning, a vaginal smear was conducted to confirm coitus after the animals were confined overnight. The first day of pregnancy was determined to be the day on which sperm was detected in the vaginal smear. Pregnant females were randomly divided into those who gave birth naturally and those who were induced to deliver prematurely. Full-term animals were obtained through the natural, non-stimulated births of rats. A single subcutaneous injection of the antiprogestin mifepristone (1 mL, 10 mg/kg body weight, Sigma-Aldrich, USA) was administered on the 20th day of gestation to induce premature labor. The introduction of antiprogestins to pregnant rats led to labor onset within 18–24 hours [5].

Primary outcome of the study

The structural characteristics of cardiomyocytes in premature rats, as determined on day 180 of postnatal ontogenesis, were the primary outcome.

Group analysis

The study was conducted on full-term (n=4, 22 days of gestation) and premature (n=4, 21 days of gestation) sexually mature male Wistar rats. To confirm premature birth, the rats were weighed at the time of birth using an HL-100 laboratory balance (Japan).

Methods of recording outcomes

The rats were euthanized by carbon dioxide asphyxiation on the 180th day of postnatal ontogenesis. Myocardial fragments from the middle third of the lateral wall of the left ventricle were fixed in 4% paraformaldehyde (Serva, Germany) for a day, then in 1% osmium tetroxide (SPI, USA) for 3 hours. The fragments are then embedded in a mixture of Epon 812, Araldite 502, and DDSA resins (SPI, USA). Sections of 80 nm thickness were obtained using a Leica EM UC 7 ultramicrotome (Leica, Austria) and contrasted with uranyl acetate and lead citrate. The ultrastructural analysis was performed using the JEM-1400 transmission electron microscope (JEOL, Japan). The morphometric analysis of the obtained images was conducted using the ImageJ 1.48 software (NIH, USA). Electronograms of longitudinal sections of contractile cardiomyocytes were used to determine the relative areas of the nucleus, cytoplasm, myofibrils, and mitochondria. Morphometric analysis was conducted on a minimum of five cardiomyocytes from each sample of all animals included in the investigation.

Ethical review

The study was conducted in accordance with the Federal Law of the Russian Federation “On the Protection of Animals from Cruel Treatment” dated January 1, 1997. The research protocol has been approved by the local ethics committee of the Siberian State Medical University of the Ministry of Health of Russia (No. 8475/1, dated November 30, 2020).

Statistical analysis

The quantitative indicators were analyzed using the SPSS 16.0 program (IBM, USA). Descriptive statistics data are presented as medians and quartiles (Me [Q1; Q3]). The normality of the distribution was verified using the Shapiro-Wilk test. We used the Mann-Whitney criterion to identify differences between groups due to the non-normal distribution. The statistical significance of differences was established at a level of p < 0.05.

Results

Study participants

At the time of birth, the weight of premature male rats was 4.3 g (4.1; 4.6), which is significantly (p=0.000) lower than the weight of full-term male rats at 6.0 g (5.7; 6.2).

Main study results

The ultrastructural structure of cardiomyocytes in premature and full-term rats on day 180 of the postnatal period was detected to be comparable (Fig. 1).

Fig. 1. Myocardium of the left ventricle of term (a) and preterm (b) male rats, 180 days of postnatal ontogenesis. Uranyl acetate and lead citrate, ×20 000 (a), ×12 000 (b). Perinuclear swelling of the cytoplasm (*) of a cardiomyocyte of a preterm animal.

Рис. 1. Миокард левого желудочка доношенных (а) и недоношенных (b) самцов крыс на 180-е сутки постнатального онтогенеза. Окрашивание уранил ацетатом и цитратом свинца, ×20 000 (а), ×12 000 (b). Звёздочкой (*) отмечено перинуклеарное набухание цитоплазмы кардиомиоцита у недоношенного животного.

The myocardium was composed of cardiomyocytes with two oval-shaped nuclei that were centrally located and dominated by euchromatin. However, the relative area of cardiomyocyte nuclei in premature rats was significantly lower, while the cytoplasm was higher than that of full-term animals (Table 1). The observed differences were linked to the development of perinuclear swelling of the cytoplasm in the cardiomyocytes of premature rats (Fig. 1, b).

The myofibril bundles in the cardiomyocytes of premature rats are oriented appropriately. The relative area of myofibrils does not differ from that in full-term rats (see Table 1). Myofibril thinning was observed in certain cardiomyocytes of premature rats. (Fig. 2, a).

Table 1. Relative area of rat cardiomyocytes structural elements

Таблица 1. Относительная площадь структурных элементов кардиомиоцитов крыс

* statistically significant differences from the index of term rats.

* статистически значимые отличия от показателя доношенных крыс.

Fig. 2. A fragment of the cytoplasm of a cardiomyocyte from preterm male rats (a, b) 180 days of postnatal ontogenesis. Uranyl acetate and lead citrate, ×25 000 (a, b). Mitochondrial damage (arrows). Mitochondria with concentric cristae (arrowshead ). Thinning of myofibrils (*).

Рис. 2. Фрагмент цитоплазмы кардиомиоцита недоношенных самцов крыс (а, b) на 180-е сутки постнатального онтогенеза. Окрашивание уранил ацетатом и цитратом свинца, ×25 000 (а, b). Повреждение митохондрий обозначено стрелками; головки стрелок указывают на митохондрии с концентрическими кристами. Звёздочками (*) отмечено истончение миофибрилл.

Numerous mitochondria were observed to exhibit subsarcolemmal, intermyofibrillar, and perinuclear localization in the cardiomyocytes of premature rats, as observed in full-term animals. On day 180 of postnatal ontogenesis, we found no differences in the relative area of cardiomyocyte mitochondria between premature and full-term rats (Table 1). However, cardiomyocytes of premature rats exhibited mitochondria with damage to the inner or both membranes (see Fig. 2, a). Alterations were detected in mitochondria regardless of their location within the cardiomyocyte. Mitochondria with concentric cristae were found exclusively in the cardiomyocytes of premature rats (see Fig. 2, b). The cardiomyocytes of premature rats exhibited a scattered arrangement of mitochondria in the cytoplasm, while the mitochondria were arranged in clusters in the cells of full-term animals (Fig. 3).

Fig. 3. A fragment of the cytoplasm of a cardiomyocyte from term (a) and preterm (b) male rats, 180 days of postnatal ontogenesis. Uranyl acetate and lead citrate, ×18 000 (a), ×30 000 (b). Dissociation of clasters of mitochondria in the cytoplasm of a cardiomyocyte of a preterm animal (*).

Рис. 3. Фрагмент цитоплазмы кардиомиоцита доношенных (а) и недоношенных (b) самцов крыс на 180-е сутки постнатального онтогенеза. Окрашивание уранил ацетатом и цитратом свинца, ×18 000 (а), ×30 000 (b). Звёздочками (*) отмечена диссоциация кластеров митохондрий в цитоплазме кардиомиоцита недоношенного животного.

On day 180 of postnatal ontogenesis, cardiomyocytes did not exhibit any ultrastructural changes in the structure of the sarcoplasmic reticulum and T-tubules, as well as intercellular contacts.

Discussion

Summary of the main study result

Cardiomyocytes did not exhibit any ultrastructural alterations in the structure of the sarcoplasmic reticulum and T-tubules, as well as intercellular contacts, on day 180 of postnatal ontogenesis.

Discussion of the main study results

To date, ultrastructural myocardial analysis has been conducted exclusively on premature infants who died in the neonatal period [6, 7], whereas the long-term effects of premature birth on the structural elements of the human cardiomyocyte have not been described. Compared to full-term infants, premature infants exhibited a lower degree of cardiomyocyte differentiation as well as signs of cardiomyocyte damage, including perinuclear edema and mitochondrial membrane destruction [6, 7]. In the remote postnatal period of ontogenesis, it would be intriguing to ascertain whether premature birth influences the ultrastructure of cardiomyocytes.

To accomplish this, individual experimental tests examining the cardiomyocyte ultrastructure were performed in animals that were modeled to simulate prematurity. Researchers adopted a model that maintained full-term rats during the neonatal period under hyperoxic conditions, to establish the prematurity model [8]. This study was conducted on male rats born 24 hours prematurely, since we hypothesized that the phenomenon of prematurity primarily involves the birth of an organism with structurally and functionally immature organs, which cannot be solely attributed to the effects of hyperoxia.

We identified structural and functional alterations in the cardiomyocytes of premature animals in the late postnatal period of ontogenesis. In premature animals, the cardiomyocytes exhibit mitochondrial membrane destruction, dissociation of mitochondrial clusters, and concentric organization of mitochondrial cristae. These changes lead to a disruption of energy production in cells, the decoupling of oxidation and phosphorylation processes, as well as the production of reactive oxygen species. In the late postnatal period, K.N. Goss et al. [8] demonstrated a decrease in the activity of superoxide dismutase 1 and 2 in rat myocardium modeled for prematurity (neonatal hyperoxia). Increased generation of reactive oxygen species, accompanied by decreased activity of antioxidant enzymes, may be the cause of pronounced oxidative stress.

The underlying causes for the appearance of mitochondria with a concentric arrangement of cristae are incompletely understood. However, it has been documented that such a spatial organization of cristae occurs when the mitochondrial or nuclear genome is damaged, including by oxidative stress [9, 10]. The spatial organization of cristae is facilitated by inner mitochondrial membrane proteins and phospholipids. Genomic damage may lead to dysfunction of the structural proteins and enzymes responsible for the production of mitochondrial membrane phospholipids [11]. The mitochondrial genome in cardiomyocytes in the late postnatal period of ontogenesis (1 year) has been demonstrated to be significantly damaged in a prematurity model that involves maintaining full-term rats in hyperoxic conditions during the neonatal period [8]. Damage to the mitochondrial membrane phospholipoids, in particular cardiolipin, which plays a critical role in the spatial organization of cristae, may be caused by the direct action of reactive oxygen species [12, 13]. Furthermore, hypoxia can disrupt the production of cardiolipin [14]. A disruption in the quantity or structure of cardiolipin can lead to the formation of concentric cristae within the mitochondria [11]. The concentric organization of mitochondrial cristae, according to A. Vincent et al. [10], causes a decline in their energy production efficiency.

In normal cardiomyocytes, mitochondria organize into structurally functional clusters. The coordinated functioning of mitochondria within a cluster is due to the presence of specific protein inter-mitochondrial contacts [15]. The mitochondria in the cardiomyocytes of premature rats are dispersed and do not establish inter-mitochondrial contacts. Thus, it can be concluded that there is functional discoordination among cardiomyocyte mitochondria in premature animals. Mitochondrial damage is one of the factors in the dissociation of mitochondria from the cluster. Additionally, it is recognized that the cluster does not encompass newly formed and dividing mitochondria [15].

The myofibril thinning and swelling of the perinuclear cytoplasm in the cardiomyocytes of premature animals may be a result of both energy deficiency and damage caused by reactive oxygen species.

Therefore, in the late postnatal period in prematurely born rats as well as in premature newborns [6, 7], swelling of the cytoplasm and mitochondrial damage are observed in cardiomyocytes. It can thus be inferred that premature birth leads to prolonged stereotypical disturbances in the structural elements of cardiomyocytes. Notably, individuals who are born prematurely exhibit signs of impaired energy metabolism, even in the late postnatal period. At the age of 25–26, monocytes of individuals born prematurely exhibited an increase in mitochondrial oxidative activity, but with decreased efficiency of adenosine triphosphate production [16].

Study limitations

The study’s limitation is the small sample size. In the future, additional studies comparing the observed structural disorders with the results of functional studies that have examined the activity and efficiency of oxidative phosphorylation in the mitochondria of cardiomyocytes in premature rats are planned.

Conclusions

Premature birth has a prolonged adverse effect on cardiomyocyte ultrastructure. The resulting structural alterations disrupt energy production in the cardiomyocytes of premature rats in the late postnatal period of ontogenesis.

Additional information

Funding source. This work was supported by the Russian Science Foundation (project No. 24-25-00015).

Competing interests. The authors declare that they have no competing interests.

Authors’ contribution. All authors made a substantial contribution to the conception of the work, acquisition, analysis, interpretation of data for the work, drafting and revising the work, final approval of the version to be published and agree to be accountable for all aspects of the work. V.V. Ivanovа ― experimental procedures, collection and analysis of literary sources, writing the text and editing the article; I.V. Milto ― experimental procedures, editing the article.

Acknowledgments. We thank the leading engineer of the Center for collective use of the Institute of Physics and Mathematics of the Russian Academy of Sciences “NANOTECH”, Candidate of Physical and Mathematical Sciences Andrey A. Miller and the head of the Microscopic Research Group of the Institute of Physical and Mathematical Physics of the SB RAS, Doctor of Biological Sciences, Professor Elena I. Ryabchikova for the opportunity to conduct electron microscopic research on a modern equipment.

Дополнительная информация

Источник финансирования. Исследование проведено при поддержке Российского научного фонда (проект № 24-25-00015).

Конфликт интересов. Авторы декларируют отсутствие явных и потенциальных конфликтов интересов, связанных с публикацией настоящей статьи.

Вклад авторов. Все авторы подтверждают соответствие своего авторства международным критериям ICMJE (все авторы внесли существенный вклад в разработку концепции, проведение исследования и подготовку статьи, прочли и одобрили финальную версию перед публикацией). Наибольший вклад распределён следующим образом: В.В. Иванова ― проведение эксперимента, сбор и анализ литературных источников, написание текста и редактирование статьи; И.В. Мильто ― проведение эксперимента, редактирование статьи.

Благодарности. Авторы выражают признательность ведущему инженеру ЦКП ИФПМ СО РАН «НАНОТЕХ», канд. физ.-мат. наук Миллеру Андрею Александровичу и заведующей Группой микроскопических исследований ИХБФМ СО РАН, д-ру биол. наук, профессору Рябчиковой Елене Ивановне за возможность проведения электронно-микроскопического исследования на современном оборудовании.

作者简介

Vera V. Ivanova

编辑信件的主要联系方式.
Email: ivvera92@rambler.ru
ORCID iD: 0000-0002-2530-1112
SPIN 代码: 9476-3329

Cand. Sci. (Biology)

Ivan V. Milto

Email: milto_bio@mail.ru
ORCID iD: 0000-0002-9764-4392
SPIN 代码: 4919-2033

Dr. Sci. (Biology), Assistant Professor

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2. Fig. 1. Myocardium of the left ventricle of term (a) and preterm (b) male rats, 180 days of postnatal ontogenesis. Uranyl acetate and lead citrate, ×20 000 (a), ×12 000 (b). Perinuclear swelling of the cytoplasm (*) of a cardiomyocyte of a preterm animal.

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3. Fig. 2. A fragment of the cytoplasm of a cardiomyocyte from preterm male rats (a, b) 180 days of postnatal ontogenesis. Uranyl acetate and lead citrate, ×25 000 (a, b). Mitochondrial damage (arrows). Mitochondria with concentric cristae (arrowshead ). Thinning of myofibrils (*).

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4. Fig. 3. A fragment of the cytoplasm of a cardiomyocyte from term (a) and preterm (b) male rats, 180 days of postnatal ontogenesis. Uranyl acetate and lead citrate, ×18 000 (a), ×30 000 (b). Dissociation of clasters of mitochondria in the cytoplasm of a cardiomyocyte of a preterm animal (*).

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