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Cardiology Research

Original Article

Volume 13, Number 6, December 2022, pages 339-356

Modeling Doxorubicin-Induced Cardiomyopathy With Fibrotic Myocardial Damage in Wistar Rats

Anthracyclines belong to the group of substances, which have both antimicrobial and antitumor activity. These drugs are commonly used for malignant tumors of various origins, such as oncohematological, soft tissue sarcomas, carcinomas, and other solid tumors [1-3]. Daunorubicin was the first anthracycline to be discovered in 1960s; it is naturally produced by an actinobacteria species called Streptomyces peucetius [4, 5]. Nowadays, the most clinically important anthracyclines are doxorubicin, daunorubicin, epirubicin, and idarubicin [5].

Side effects after anthracyclines chemotherapy include cardiovascular disease. Development of heart failure causes a considerable deterioration in the quality of life and expectancy of those patients who were previously successfully treated for malignant neoplasms. Doxorubicin is known to induce myocardial cell death through exploiting a variety of mechanisms: apoptosis, autophagy, necroptosis, ferroptosis, and pyroptosis [6-10].

To date, a complete understanding of the mechanisms of anthracycline (doxorubicin) cardiotoxicity development has not been gained. Doxorubicin is able to block the nuclear enzyme topoisomerase II, which controls the topological state of DNA in the cell. Anthracyclines are easily captured by cells and localized in the nucleus [11]. The intercalation function suppresses the synthesis of DNA and RNA in highly replicating cells (anti-cancer effect) and resting cells (cardiomyocytes), subsequently blocking the processes of transcription and replication. Anthracyclines, as DNA intercalators, form a stable ternary anthracycline-DNA-topoisomerase IIβ complex, thus “poisoning” the enzyme and preventing re-ligation of double-stranded DNA breaks. Topoisomerase II-mediated DNA damage subsequently stops proliferation and activates the DNA repair mechanism. When the repair process fails, the lesions initiate a programmed cell death [12-14]. Moreover, doxorubicin is able to form reactive oxygen species (ROS). The quinone fragment of anthracyclines can undergo redox reactions with formation of an excess of ROS in the presence of oxidoreductive enzymes, such as cytochrome P450 reductase, NADH dehydrogenase, and xanthine oxidase [15-17]. Conversion of quinone to semiquinone produces free radicals which actively react with oxygen to generate superoxides, hydroxyl radicals and peroxides [2, 3, 18, 19]. Additionally, the availability of cellular iron catalyzes redox reactions and generates ROS. Excessive ROS that cannot be utilized contribute to oxidative stress, DNA damage and lipid peroxidation, thereby causing apoptosis and necrosis [20-23]. All mechanisms ultimately lead to the cardiomyocyte death and proliferation of connective tissue, and cicatricial (fibrotic) changes in the cardiac muscle. However, a number of clinical trials have demonstrated that patients with cardiotoxicity revealed during anthracyclines therapy also have extensive fibrotic changes in the heart muscle in the long term, which worsen the prognoses of such patients [24].

At the moment, the clinic has established the following figures for the incidence of congestive heart failure against the background of the use of appropriate doses of doxorubicin: 4.7% (400 mg/m²), 26% (550 mg/m²) and 48% (700 mg/m²) [14, 25]. Therefore, the cumulative lifetime exposure to doxorubicin is limited to 400 - 450 mg/m² in order to reduce the incidence of heart failure to less than 5%. At the same time, different people have differences in tolerance to doxorubicin. Risk factors that influence the degree of anthracycline-induced cardiac injury include genetic variation [26], age (younger or older age groups), previous treatment with cardiotoxic drugs, and a history of heart disease [27]. Children are particularly at risk due to anthracycline activity, which can compromise the development of an immature heart. The exact mechanism for the development of cardiotoxicity remains unclear, due to the poorly understood mechanisms of anthracyclines action.

Researching genetic mechanisms of fibrosis formation in the doxorubicin-induced cardiomyopathy development remains relevant. The following is being researched: transforming growth factor (TGF)-β signaling pathway and its effect on COL type I, II and III gene expression, interaction of TGF-β with fibroblast growth factors (FGF) [28, 29], contribution of inflammatory tumor necrosis factor (TNF)-α [30, 31], participation of alpha-smooth muscle actin (α-SMA) [32], the role of matrix metalloproteinases (MMP) and their inhibitors, tissue inhibitor of metalloprotease (TIMP) [33], and factors, which additionally regulate fibroblast activity, such as endothelin (ET)-1, adrenaline, and angiotensin 2 [34].

Despite the numerous researches about chronic doxorubicin-induced cardiomyopathy modeling, the attention was mainly focused on the clinical criteria of cardiac dysfunction (decreased ejection fraction, increased markers of myocardial damage) and such histological patterns as structural damage in the myocardium and tissue edema [15]. The effects of free radicals due to the activation of oxidative stress during anthracyclines therapy not only damage the heart tissue, but also involve the kidneys in a pathological process with subsequent impairment of their function [35]. Doxorubicin has a harmful effect on the glomeruli, leading to severe proteinuria [36, 37]. Affected by a persistent systemic inflammation and activation of oxidative stress proteinuria [38], in its turn, leads to fibrotic changes in the kidneys, which can further significantly impair their function [39]. Thus, it is observed a close relationship between pathophysiological changes in the renal function and such changes in the cardiac function during the development of heart failure, and the peculiarities of modern cardioprotective therapy against the background of doxorubicin treatment, which includes therapy with drugs belonging to the group of renin-angiotensin-aldosterone system (RAAS) blockers. The modern chronic doxorubicin-induced cardiomyopathy experimental model should consider not only characteristic changes in the myocardial function, including an assessment of the degree of fibrosis, but also the background state of the renal excretory system.

To date, a lot of data have been published to be devoted to the doxorubicin-induced cardiomyopathy modeling using various animal species [15, 40-46]. Nevertheless, taking into account the lack of an unambiguous understanding of the mechanisms of the fibrotic changes formation in the cardiac muscle under the influence of doxorubicin, consequently, a relevant experimental model of chronic doxorubicin-induced cardiomyopathy with fibrotic lesions of the heart muscle is in great demand. Creating a relevant model that meets modern conditions will allow developing new pathogenetical approaches to treatment and prevention, to improve the life quality and expectancy of patients undergoing chemotherapy.

The study was aimed to create an acute and chronic doxorubicin-induced cardiomyopathy with fibrous lesion of the left ventricle and delayed development of diastolic dysfunction in Wistar rats. Furthermore, the task to determine the main molecular markers involved in fibrous tissue formation in the chronic doxorubicin-induced cardiomyopathy development.

Doxorubicin-LENS^®, 50 mg (VEROPHARM, LLC, Russia) was used to create the model. The drug was prepared for administration to animals immediately before use (ex tempore). Doses of the drug were determined empirically, taking into account literature data. The dose of the drug calculated for each animal was diluted with saline. During the experiment, the animals were periodically weighed and the doses of the drug were adjusted.

The study in animal protocol was approved by a bioethical commission of the Centre for Experimental Biomodeling, Institute of Experimental Medicine, Almazov National Medical Research Centre, Ministry of Health of the Russian Federation (No. 20-09ПЗ#V2, date of approval: 25.06.2020). The study was conducted according to the guidelines of the Declaration of Helsinki.

The development of models was carried out in two successive stages: first stage (creation of acute doxorubicin-induced cardiomyopathy) - 35 male Wistar SPF rats weighing 393 ± 38 g (5 months); second stage (creation of chronic doxorubicin-induced cardiomyopathy) - 40 male Wistar SPF rats weighing 260 ± 19 g (3 months). In the first stage, rats with a greater weight were involved, due to the stronger effect of the chemotherapy drug. Animals were housed in a barrier-type vivarium under standardized conditions for animal facilities: 12-h light/dark cycle with food and water ad libitum. Animals were randomly divided into groups with a certain frequency of intraperitoneally administration and a certain dose of doxorubicin (Table 1).

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Table 1. Brief Description of the Experimental Groups

At the first stage of the study, echocardiography (ECHO) was recorded before the start of the chemotherapeutic agent injection and 2 days after the end of the injection. The next day, under isoflurane anesthesia (5% isoflurane-98% oxygen), vital organs, heart and kidneys, were taken. Before cardiac arrest, 10% KCl solution was injected into the left ventricle until cardiac arrest was completed in the diastole phase.

The second stage of the study included four discrete points: starting point (indicates the phase before the doxorubicin injection), 0 point (demonstrates the completion of the doxorubicin injection), first month and second month (Fig. 1). At each point, ECHO was performed and blood was taken from the retroorbital sinus for hematological analysis. Intravital blood samples, which were taken from the retroorbital sinus (0.25 mL), were performed under isoflurane anesthesia. In the subsequent stage, autopsy was performed on the animal under xylosin zolityl anesthesia (10 mg/kg xylazine + 25 mg/kg zoletil, intramuscularly), and the heart and the kidneys were removed for histological analysis. During the procedure, 10% KCl solution was injected into the left ventricle as long as the cardiac arrest happened. After removal of the heart from the chest cavity, the heart apex was cut off and subjected to deep freezing (-80 °C) for further performing of RT-qPCR.

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Figure 1. Experiment scheme. *Weighing points. IP: intraperitoneally.

From the starting point until 0 point, the animals were weighed every 2 days, then once a week (Fig. 1). In order to assess the change in the body weight, an AND DX 1200 WP electronic laboratory scale (Japan) with an accuracy of 0.01 g was used.

Before echocardiography, the animal was anesthetized using an inhalation mixture of 1.7% isoflurane-98% oxygen. The echocardiographic images were obtained with heart rate stabilized at about 400 beats per minute. Echocardiogram was recorded using The Vevo^® 2100 (VisualSonics Inc., Toronto, Canada). All parameters were taken from the right parasternal short axis. The analysis of the myocardium functioning parameters was carried out in the M mode. Left ventricular end-diastolic internal diameter at Q wave (LVIDd), left ventricular end-systolic internal diameter at T wave (LVIDs), posterior wall thickness at diastole (LVPW), and anterior wall thickness at diastole (IVS) were measured. Another parameter was calculated as follows: left ventricular short axis fractional shortening FS (%) = (LVIDd - LVIDs)/LVIDd × 100.

The heart and the kidneys were excised and fixed in 10% formaldehyde solution for 72 h. The tissue samples were encased in paraffin. In order to study the morphological changes, 3-µm thick cross-sections were made using rotary microtome Accu-Cut SRM (Sakura, Japan). The sections were stained by hematoxylin and eosin, dehydrated and enclosed in a synthetic mounting medium (Biovitrum, St. Petersburg, Russia). Morphometric studies were carried out on micrographs of the samples stained by Mallori using a Nikon Eclips 400i microscope (Nikon, Tokyo, Japan) and NIS Elements 4.3 Br software (Nikon, Tokyo, Japan). For each animal, there were five randomly selected fields of view on each histological section. Five photographs of the left ventricular (LV) of the cardiac muscle were taken at × 400 magnification. In each micrograph, the amount of collagen-stained blue was counted and expressed as a percentage of the total section area using Photoshop CS6 (Adobe Inc., California, USA). Graphically, the data were expressed as a range chart.

A general blood test was performed using an Abacus junior Vet (Diatron, Budapest, Hungary) veterinary hematological analyzer. Quantitative parameters were evaluated. The following blood ones were determined: white blood cells (WBCs, g/L); leukocyte fraction: granulocytes (Gr, %), lymphocytes (Lym, 10⁹ g/L), monocytes (Mi, %), red blood cells (RBCs, × 10¹²/L), hemoglobin (Hb, g/L); erythrocyte indices: mean concentration hemoglobin (MCH, pg), mean corpuscular hemoglobin concentration (MCHC, g/L), mean corpuscular volume (MCV, fL), hematocrit (Hct, %), and platelets (Plt, × 10⁹/L).

The total RNA was extracted from deep frozen heart apex samples using ExtractRNA™ solution and CleanRNA Standard™ Kit (Evrogen JSC, Russia) according to the manufacturer’s protocols. Reverse transcription (RT) and consequent quantitative real-time PCR (qPCR) were performed using OneTube RT-PCR SYBR™ Kit (Evrogen, JSC, Russia) following the manufacturer’s instructions. RT-qPCR was performed by a Bio-Rad CFX96 thermocycler (Bio-Rad, Hercules, CA). The parameters for RT-qPCR were set as follows: initial heating at 95 °C for 1 min and then followed by 40 cycles with denaturation at 95 °C for 15 s, annealing at 60 °C for 20 s, and extension at 72 °C for 60 s. Relative expressions of 13 genes of interest and four housekeeping genes were calculated by the 2^-ΔΔCt method and normalized by GAPDH, α-tubulin, β-actin and TBP. The primer sequences are listed in Table 2.

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Table 2. Lists of Primer Sequences Used for RT-qPCR

All calculations were performed with GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA) software for Windows (Microsoft Inc., USA). The Shapiro-Wilk test was used to detect the normal distribution. An unpaired, non-parametric Kruskal-Wallis test with Dunn’s test was used to compare between several independent groups, since the data obtained (ECHO and hematological values) are non-normally distributed. The Wilcoxon test was used for pairwise comparison of dependent values of the non-normal distribution (ECHO data, *dynamics compared to the initial value). An unpaired, non-parametric Kruskal-Wallis test with Dunn’s test was used to compare the body weight of animals and assess the histological differences between different groups. The values in the groups were processed using nonparametric statistics (median and 25th and 75th percentiles (Me (25-75%)). One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used for statistical analysis of RT-qPCR data. The values are shown as mean ± standard deviation (SD). The differences were considered significant at P < 0.05.

There was a 100% death of animals observed against the background of the injection of the drug at a dose of 25 mg/kg, while when using a lower dose (20.4 mg/kg), the death of animals was 30%. Mortality in the control and DOX-15 groups animals was not observed. The decrease in body weight on the background of doxorubicin administration was: 68 ± 14.6 g of the DOX-25 group of animals; 58.8 ± 12.2 g of the DOX-20.4 group of animals; 56.3 ± 17.4 g of the DOX-15 group of animals.

Due to the extremely severe physical condition such as substantial weight loss over 20% in a short period of time, abundant discharge of porphyrin, decreased physical activity, alteration of the consistency of feces to jelly-like and high mortality, ECHO was not possible in animals receiving 25 and 20.4 mg/kg cumulative doses of the drug. There were a significant decrease in the FS (before: 54.5±5.9%; after: 37.5±5.5%; ***P ≤ 0.001) and an increase in the LVIDs (before: 3.1 ± 0.53 mm; after: 4.1 ± 0.37 mm; ***P ≤ 0.001) on the 13th day after the start of doxorubicin injection at a dose of 15 mg/kg.

The histopathological picture of the heart muscle of the left ventricle revealed capillary plethora and dilated vessels in all animals treated with doxorubicin, the most pronounced in animals receiving the maximum cumulative dose (25 mg/kg). They are also characterized by a partial loss of myofibrils of cardiomyocytes (Fig. 2). The picture of damage to the tissues of the kidneys was characterized by hemorrhage in the medulla of the kidney, compressed and deformed nephrons. Venous plethora of the cortical and medulla of the kidneys, moderate atrophy of the tubules (decrease in the height of epithelial cells and widening of the lumen of the tubules) and necrosis of individual epitheliocytes were revealed in animals receiving 25 mg/kg of the drug (Fig. 3).

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Figure 2. Representative images of rat’s left cardiac ventricle of the control, DOX-25, DOX-20.4, and DOX-15 groups. Stained by hematoxylin and eosin, × 50.

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Figure 3. Representative images of rat’s kidney of the control, DOX-25, DOX-20.4, and DOX-15 groups (stained by hematoxylin and eosin, × 25 (control group); × 50 (DOX-25, DOX-20.4, and DOX-15 groups)).

Thus, the use of 20 and 25 mg/kg of doxorubicin ensures the development of acute cardiotoxic effects, manifested in a pronounced deterioration in the physical and clinical condition of the animals. A dose of 15 mg/kg of the drug was used to model chronic doxorubicin-induced cardiomyopathy.

There was a 30% death rate noted in animals 45 days after the last injection of the drug when using the maximum cumulative dose (15 mg/kg). There were no deaths in the other groups.

The dynamics of body weight of animals in all experimental groups is shown in Figure 4. A gradual increase in body weight was observed during the entire experiment in the control group of animals and in the DOX-5 group. Weight loss was observed during injections in the surviving animals using the maximum cumulative dose (DOX-15). In the DOX-15 group of animals, a rapid deterioration in the clinical state characterized by substantial weight loss, abundant discharge of porphyrin, and decreased physical activity of the animals was observed, which led to their earlier withdrawal from the experiment.

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Figure 4. Dynamics of body weight in animals of the DOX-15, DOX-10, and DOX-5 groups. *Significant differences between the DOX-15 group of animals versus the DOX-10, DOX-5, and control groups (**P < 0.01, ***P < 0.001).

No changes in the echocardiography parameters were observed during the experiment in the control group animals.

There was a significant increase in the LVIDs and LVIDd in animals after the injection of all doses (P < 0.05, compared to the initial value, compared to the control group). Besides, a significant decrease in the FS was observed in all experimental groups (Fig. 5). The most substantial decrease in FS (41% of the initial value) was observed in animals after the injection of the maximum cumulative dose of the drug (DOX-15). Whereas for the group of animals receiving 10 mg/kg, a decrease in FS by 15% was noted, and 14% in the DOX-5 group of animals.

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Figure 5. LVIDs, LVIDd and FS dynamics of animals of the DOX-15, DOX-10, and DOX-5 groups. *Dynamics compared to the initial value (e.g., in the DOX-15 group; *P < 0.05, **P < 0.01, ***P < 0.001). Δ: dynamics compared to the highest dose at each point of the experiment (e.g., in first month: DOX-5, control vs. DOX-15; Δ: P < 0.05, ΔΔ: P < 0.01, ΔΔΔ: P < 0.001). LVIDs: left ventricular end-systolic internal diameter; LVIDd: left ventricular end-diastolic internal diameter; FS: fractional shortening.

A slight increase in the dimensions of the anterior (IVS) and posterior (LVPW) walls thickness in left ventricular diastole was revealed in the animals receiving 10 and 5 mg/kg cumulative doses (Fig. 6). A significant increase in both IVS and LVPW was found between the animals of the DOX-10 and DOX-5 groups compared to the DOX-15 group (P < 0.05). Depletion of both the anterior and posterior walls thickness of the left ventricle was observed during the drug injection in animals receiving a cumulative dose of 15 mg/kg of doxorubicin by the end of the second month.

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Figure 6. IVS and LVPW dynamics of animals of the DOX-15, DOX-10, and DOX-5 groups. *Dynamics compared to the initial value (*P < 0.05, **P < 0.01, ***P < 0.001). Δ: dynamics compared to the highest dose at each point of the experiment (e.g., in the second month: DOX-10, DOX-5 vs. DOX-15; Δ: P < 0.05, ΔΔ: P < 0.01). IVS: the dimensions of the anterior wall thickness; LVPW: the dimensions of the posterior wall thickness.

The results of a general blood test showed a significant decrease after the drug injection (0 point) in such indicators as WBC, Lym, GR, Mi, Hb, MCH, and MCHC in animals of the DOX-15 and DOX-10 groups compared to the control group (Table 3). It can be assumed that these changes are associated with the acute toxic effect of anthracycline in the form of inhibition of hematopoiesis in animals that received the highest doses. After a month, the values of hematological parameters returned to normal ones, which indicates the reversibility of this effect.

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Table 3. Quantitative Indicators of Hematological Analysis of Animals of the DOX-15, DOX-10, DOX-5 Groups

Histology of the left cardiac ventricle tissue in animals with chronic doxorubicin-induced cardiomyopathy revealed foci of cardiomyocytes with pronounced vacuolization of the cytoplasm and hemorrhages at the sites of necrosis. The heart muscle of animals receiving 10 and 5 mg/kg was less damaged, but at the same time, there was also a partial loss of nuclei, vacuolated cytoplasm and necrosis of contraction bands, which is a sign of focal necrosis at the stage of cell decay with destruction of the vascular wall and minor hemorrhage (Fig. 7). Stained by Mallori of the left cardiac ventricle revealed an increase in the percentage occupied by collagen in animals receiving 15 and 10 mg/kg of doxorubicin compared to the control group (DOX-15: 11.42% (9.920; 13.61); DOX-10: 7.77% (4.35; 10.84); DOX-5: 5.42% (4.568; 6.408); control: 3.23% (2.465; 3.620)) (Figs. 8, 9). The histopathological picture of the kidney tissue showed the presence of segmental glomerulosclerosis and tubular epithelial necrosis most pronounced in animals treated with 15 mg/kg doxorubicin (Fig. 10).

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Figure 7. (a) Representative images of rat’s left cardiac ventricle of the control group; × 50; stained by hematoxylin and eosin. (b, c) Representative images of rat’s left cardiac ventricle of the DOX-15 group; stained by hematoxylin and eosin. (b) Dissociation of myocardial fibers is seen, forming cavities filled with blood (arrow); × 50. (c) Asterisks show vacuolization of the cytoplasm of cardiomyocytes, black arrows show nuclei of cardiocytes that undergo karyolysis, white arrows show hypertrophied fibers; × 25. (d) Representative images of rat’s left cardiac ventricle of the DOX-10 group; × 25; stained by hematoxylin and eosin. Anisonucleosis, medium and large cell vacuolization of the cytoplasm (arrows). (e) Representative images of rat’s left cardiac ventricle of the DOX-5 group; × 25; stained by hematoxylin and eosin. The arrows show vacuolization of the cytoplasm of cardiomyocytes, the absence of visible boundaries between individual cells.

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Figure 8. Representative images of rat’s left cardiac ventricle of the control, DOX-15, DOX-10, and DOX-5 groups. Stained by Mallori, collagen is colored blue; × 25.

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Figure 9. The amount of collagen in the left cardiac ventricle with chronic doxorubicin-induced cardiomyopathy of rats of the control, DOX-15, DOX-10, and DOX-5 groups, %. Kruskal-Wallis test.

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Figure 10. (a) Rat’s kidney of the Control group; × 25; stained by hematoxylin and eosin. (b, c) Rat’s kidney of the DOX-15 group; × 25; stained by hematoxylin and eosin. (b) The arrow shows the necrosis of the tubular epithelium, the sclerosed glomerulus shows in the center. (c) Massive tubular epithelial necrosis (arrow). (d) Rat’s kidney of the DOX-10 group; × 50; stained by hematoxylin and eosin. Global glomerulosclerosis of three renal glomeruli. (e) Rat’s kidney of the DOX-5 group; × 25; stained by hematoxylin and eosin. The black arrow shows necrosis of individual tubular epithelial cells. White arrows show thickening of the basement membrane of the glomerulus. Vacuolization of the cells of the parietal leaf of the capsule (asterisk).

The disorders ultimately lead to the pronounced fibrosis development of the left ventricular of the cardiac muscle within 2 months in the animals that received 10 mg/kg of anthracycline. There is no statistically significant increase in the percentage of fibrous tissue of the cardiac muscle in rats treated with 5 mg/kg of the drug compared to the control group. Due to a progressive doxorubicin-induced cardiomyopathy development and multiple vital organs failure (extensive necrosis of hepatocytes, segmental glomerulosclerosis, necrosis of tubular epithelium) rats’ intake 15 mg/kg of doxorubicin began to lose much weight 45 days after the end of injections, which was incompatible with life. This led to the ascites development for one animal, presumably due to a severe heart failure associated with cardiomyopathy, which is also a side effect of doxorubicin. Thus, a dose of 15 mg/kg causes a pronounced cardiotoxic effect.

Two months after the end of doxorubicin administration, an RT-qPCR analysis found no changes in mRNA level of myocardial TGF-β1 in all experimental groups compared to the control group (Fig. 11). Expression of COL1A1 and COL2A1 significantly increased in the DOX-15 group (P < 0.01); there were no changes in the DOX-10 group; in the group with the minimum cumulative dose of doxorubicin (5 mg/kg), the level of mRNA of both genes significantly decreased (P < 0.001). At the same time, a downregulation of COL3A1 expression was found in all treatment groups. Animals from the DOX-10 group were characterized by downregulation of FGF2 (P < 0.05). In turn, heart tissues of rats, consuming the maximum (15 mg/kg) and minimum (5 mg/kg) doses of doxorubicin, showed an upregulation of FGF4 (P < 0.001). The downregulation of metalloproteases MMP-1 and MMP-14 expressions was found in the DOX-15 group and DOX-5 group, respectively (P < 0.001). Interestingly, MMP-2 was not affected by drug treatments in any of the groups. In contrast to the effect of MMP-1, mRNA level of TIMP-2 increased in the DOX-15 group (P < 0.01). At the same time, TIMP-1 expression decreased in the animals of the DOX-10 group (P < 0.05). Two months after the DOX-treatment, there was a significant increase in ET-1 in the group with the maximum cumulative dose (P < 0.001) and a significant decrease in animals receiving the minimum dose of the drug (P < 0.001). Expression of α-SMA was reduced dramatically in all experimental groups compared to the control group (P < 0.001). An increased mRNA level of TNF-α was specific only for the DOX-15 group animals (P < 0.001), while its expression is significantly down in the DOX-10 and DOX-5 groups (P < 0.001) (Fig. 11).

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Figure 11. Gene expression levels of fibrotic molecular markers in the model of chronic doxorubicin-induced cardiomyopathy. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control. COL1A1, COL2A1, COL3A1: collagen, type I, II, III; ET-1: endothelin-1; FGF: fibroblast growth factors; MMP: matrix metalloproteinases; TGF-β: transforming growth factor-β; TIMP: tissue inhibitor of metalloprotease; TNF-α: tumor necrosis factor; α-SMA: alpha-smooth muscle actin.

This study is devoted to the development and verification of the acute and chronic model of doxorubicin-induced cardiomyopathy with fibrous myocardial damage and delayed development of diastolic dysfunction. The doxorubicin-induced cardiomyopathy pathogenesis is characterized by: 1) active accumulation of the drug in cardiomyocytes, which leads to excessive ROS generation, 2) development of inflammatory processes, 3) oxidative stress, which, in turn, disrupts the functioning of mitochondria, the sarcoplasmic reticulum, the balance of calcium and iron ions in the cell, 4) blocks the work of topoisomerase IIβ, and, ultimately, 5) causes the death of cardiomyocytes. The death of cardiomyocytes leads to their gradual replacement with fibrous tissue with diastolic dysfunction development.

To date, there has not been a final understanding of the mechanism for the anthracycline cardiomyopathy development, which makes it difficult to develop new methods of prevention and treatment despite the abundance of data on the various mechanisms involved in the doxorubicin-induced cardiotoxicity implementation. At the same time, researchers are actively trying to develop new models that provide a cardioprotective effect. For example, He et al demonstrated that repeated remote ischemic preconditioning eliminated the effects of doxorubicin-induced cardiotoxicity: it reduced the decline in cardiac function, weakened the formation of cardiac fibrosis, and reduced the processes development in cardiomyocytes [47]. Many modern works are aimed at stimulating sirtuins through various signaling pathways that have anti-inflammatory, antioxidant, anti-apoptotic effects on the myocardium [23, 48-50].

The study was performed on Wistar rats. The choice of this type of animals was motivated by a large number of published works devoted to doxorubicin cardiotoxic effects modeling in rats [40, 43, 51, 52]. In order to level the possibility of the influence of hormonal changes on the results obtained in the modeling of pathological processes, sexually mature males were used in this work.

The data obtained from the study indicate that a six-fold injection of 20 mg/kg doxorubicin provides manifestations of pronounced cardiotoxicity, manifested by a pronounced decrease in body weight of animals up to 20% in a short period of time, severe damage to both the cardiac muscle of the left ventricle and the renal tissue, high mortality at the end of the experiment. Whereas the results of animals treated with 15 mg/kg of the drug showed a high survival rate. As a result, this dose was chosen as a “standard” for the chronic model of doxorubicin-induced cardiomyopathy development.

As the main criteria for verification of the chronic model of doxorubicin-induced cardiomyopathy with fibrous myocardial damage and delayed development of diastolic dysfunction, the authors used the following: the increase in the percentage of collagen; the degree of decrease of FS, which characterizes the pumping ability of the heart; clinical blood test, reflecting the toxic effect of the drug on hematopoiesis. To confirm fibrotic changes identified by histological analysis, an additional genetic analysis was carried out for the differential expression of fibrosis marker genes: TGF-β, COL1A1, COL2A1, COL3A1, FGF2, FGF4, α-SMA, TNF-α, ET-1, MMP-1, 2, 14, TIMP-1, 2.

To fulfill the task, a range of doxorubicin doses of 5 - 15 mg/kg was used, and the choice of doses was based on literature data [15]. The observation time after reaching the cumulative dose of the drug was 2 months, which is sufficient for the development of delayed effects of doxorubicin, myocardial fibrosis and diastolic dysfunction [53].

The results obtained showed that the administration of doxorubicin in doses of 10 and 15 mg/kg affected the physical condition and weight gain of the animals. A dose of 15 mg/kg of the drug caused weight loss against the background of a pronounced deterioration in the clinical condition of the animals, while the administration of 10 mg/kg of doxorubicin stopped the body weight gain, which could also be a manifestation of the toxic effect of doxorubicin. Moreover, with the administration of the drug in doses of 10 and 15 mg/kg, hematopoiesis inhibition was observed, which also indicates the toxic effect of doxorubicin. During the administration of all doses (5, 10 and 15 mg/kg), there was a significant decrease in FS, which reflects an impairment of the myocardium diastolic function, also affected by the resulting fibrous tissue. FS is the change in left ventricular size during each heartbeat expressed as a percentage and normally should not decrease by more than 10%. It is known from literature sources that one of the indicators of subclinical cardiotoxicity development determined via echocardiography in humans is a drop in both FS and ejection fraction [10, 54]. Therefore, as per the results of echocardiography, it can be concluded that doses of 10 and 5 mg/kg doxorubicin work best for creating chronic anthracycline cardiomyopathy since they contribute to a decrease in FS, but they are not critical for the life of the animal. The presence of fibrotic changes was confirmed by histological analysis. Thus, in animals receiving 10 and 15 mg/kg, a significant increase in the percentage of collagen in the heart muscle was revealed. The histological picture of the kidneys also demonstrated the presence of a nephrotoxic effect when using cumulative anthracycline doses of 10 and 15 mg/kg.

Two months after the end of chemotherapy administration, an analysis of fibrosis molecular markers showed that mRNA level of myocardial TGF-β did not change. At the same time, an increase in COL type I and II, FGF4 in animals that had received the highest dose of doxorubicin (15 mg/kg) remained. Whereas the amount of interstitial collagenase-1 (MMP-1) was significantly reduced in animals of the DOX-15 group, which possibly correlates with TIMP2 activation. These results may indicate a temporary adjustment of the regulation of the TGF/FGF and MMP/TIMP systems during the formation of fibrous tissue against the background of the toxic effect of doxorubicin [55]. However, it was found that the excessive production of myocardial collagen type I and II (COL1A1 and COL2A1) persisted after 2 months in the DOX-15 group animals, while it did not change in the DOX-10 group animals, and it significantly decreased in the DOX-5 group animals. It can be assumed that 2 months after reaching the cumulative dose of doxorubicin, despite the absence of significant changes in TGF-b, the formation of fibrosis is not complete, and the preserved FGF4 expression may play a greater role in the development of chronic doxorubicin-induced cardiomyopathy. It is important to note a decline in type III myocardial collagen in all experimental groups. The main consequence of fibrosis is a fall in the ventricular compliance. Ventricular compliance decreases both due to an increase in the number of collagen fibers and as a result of the collagen properties variation. That is, a decrease in the content of “elastic” type III collagen and an increase in the content of “hard” type I collagen [60]. Thus, fibrosis progresses to the development of diastolic dysfunction.

It should be noted that the excess production of ET-1 persists after 2 months of observation in animals treated with 15 mg/kg of the drug, while a significant decrease in ET-1 was characteristic of animals of the DOX-5 group. A number of studies show that ET-1 can induce collagen synthesis in cardiac fibroblasts via the ETA receptor, and this effect was similar to that of TGF-β [56-58]. Our data support the role of ET-1 as a key player in cardiac fibrosis. In addition, an increase in TNF-α was revealed in the group of animals treated with 15 mg/kg of the drug, which indicates there are persisting inflammatory processes in the heart muscle. Whereas in animals of the DOX-10 and DOX-5 groups, a significant decrease in the inflammatory factor was observed 2 months after the end of its administration. This result confirms that a dose of 15 mg/kg doxorubicin is highly cardiotoxic. The fibrous factor α-SMA was reduced in all experimental groups, which may indicate that it may not be a significant marker of fibrous tissue formation in the development of chronic doxorubicin-induced cardiomyopathy [32, 59]. In general, the data obtained confirm the presence of fibrotic changes in the cardiac muscle.

The authors did not evaluate troponin and N-terminal pro B-type natriuretic peptide (NT-proBNP) levels for model verification since literature data indicate the ambiguity of the relationship between these criteria and the severity of myocardial damage under the action of doxorubicin [60-64]. A limited set of criteria was used to verify the acute model of doxorubicin-induced cardiomyopathy. These criteria are basic and sufficient to determine the optimal dose range for creating the chronic model of doxorubicin-induced cardiomyopathy.

Therefore, inflammatory processes and active synthesis of fibrous tissue that persisted after 2 months in animals treated with 15 mg/kg of doxorubicin correlate with a progressive deterioration of their clinical condition. This dose is highly cardiotoxic, which does not allow us to consider it suitable for creating chronic doxorubicin-induced cardiomyopathy. A dose of 10 mg/kg of the drug is associated with fibrotic changes and is suitable for assessing chronic cardioprotective effects. A dose of 5 mg/kg of doxorubicin does not ensure the development of the processes of formation of fibrotic changes, and, consequently, cardiotoxicity. It is necessary to note the preserved expression of ET-1, FGF4 and TNF-α, which can affect the regulation of TGF-β, myocardial collagen types I, II, III and MMP/TIMP during the formation of chronic processes against the background of chemotherapy. This molecular genetic aspect remains to be studied in more detail, since understanding the interaction of fibrotic markers with each other can help in the development of methods for the prevention and treatment of anthracycline cardiotoxicity.

The optimal cumulative dose of doxorubicin leading to clinical manifestations confirmed by echocardiographic, histological, molecular changes associated with the development of chronic doxorubicin-induced cardiomyopathy with fibrotic lesions of the left ventricular of the cardiac muscle and ensuring long-term survival of animals is 10 mg/kg doxorubicin. The model with intraperitoneal administration of 10 mg/kg doxorubicin to Wistar rats can be used to study the mechanisms of development of anthracycline cardiotoxicity as close as possible to the changes that develop clinically in those people who have undergone chemotherapy.

Acknowledgments

None to declare.

Financial Disclosure

This work was financially supported by the Ministry of Science and Higher Education of the Russian Federation (agreement No. 075-15-2020-901).

Conflict of Interest

The authors confirm that they have no conflict of interest to declare.

Informed Consent

Not applicable.

Author Contributions

EP: conceptualization, investigation, data collection, analysis, writing - original draft preparation and editing; TS: material preparation, data collection, and writing - original draft preparation; EK: writing - original draft preparation; AO: material preparation; MM: investigation; DA and RT: material preparation and data collection; YC: investigation; KL: editing; AG: editing; YT: conceptualization, editing, project administration, funding acquisition.

Data Availability

The authors declare that data supporting the findings of this study are available within the article.

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