Abstract
Over the past 70 years, there has been extensive research focused on preventing chemotherapy-related cardiovascular complications. However, the current state of cardio-oncology research has raised more questions than answers. Experimental studies often present data that are difficult to compare and, at times, contradictory. One notable limitation in translating experimental findings to clinical practice is the reliance on models that administer only one chemotherapeutic drug to experimental animals, despite the common use of multidrug cancer treatments in real clinical settings. This article aims to discuss our own experience in modeling an experimental rat model of cardiomyopathy induced by the administration of two chemotherapeutic drugs, doxorubicin (adriamycin) and cyclophosphamide (AC mode of chemotherapy) – Avagimyan A., et al model, along with a subsequent review of morphological changes based on our personal archive.
- 1.
AC – Doxorubicin (Adriamycin) + Cyclophosphamide
- 2.
CaMKII – Calcium/Calmodulin-dependent protein kinase II
- 3.
Circ-INSR – Circulating Insulin Receptor
- 4.
CVDs – Cardiovascular Diseases
- 5.
CP – Cardioprotector
- 6.
GLUT – Glucose Transporter
- 7.
HF – Heart Failure
- 8.
ICAM – Intercellular Adhesion Molecule
- 9.
IL – Interleukin
- 10.
IκBα – Inhibitor of kappa B alpha
- 11.
MMP – Matrix Metalloproteinase
- 12.
MyD88 – Myeloid Differentiation Primary Response 88
- 13.
NADP+ – Nicotinamide Adenine Dinucleotide Phosphate
- 14.
NADPH – Nicotinamide Adenine Dinucleotide Phosphate Hydrogen
- 15.
NF-κB – Nuclear Factor kappa B
- 16.
NLRP3 – Nucleotide-binding domain, leucine-rich repeat containing protein 3
- 17.
NOS – Nitric Oxide Synthase
- 18.
Nrf2 – Nuclear factor erythroid 2-related factor 2
- 19.
NT-proBNP – N-terminal pro-B-type natriuretic peptide
- 20.
PLN – Phospholamban
- 21.
RyR – Ryanodine receptor
- 22.
SERCA2a – Sarco/Endoplasmic Reticulum Calcium ATPase 2a
- 23.
Sirt3 – Sirtuin 3
- 24.
SMAD3 – Mothers Against Decapentaplegic Homolog 3
- 25.
TGF-β1 – Transforming Growth Factor beta 1
- 26.
TLR4 – Toll-like receptor 4
- 27.
TNF-α – Tumor Necrosis Factor alpha
- 28.
VCAM – Vascular Cell Adhesion Molecule
Introduction
Cardiovascular disease (CVD) and cancer are the leading causes of death in economically developed countries. , Interestingly, an increasing number of patients with malignant neoplasms die from non-cancer-related causes, with CVD being the most prevalent Notably, cancer survivors face equally dangerous complications because of tumor treatment – heart failure (HF), the pandemic of the 21st century–with a five-year mortality rate of approximately 50% Consequently, addressing chemotherapy-related cardiovascular complications has become a top priority
The severity of cardiovascular toxicity induced by cancer treatment was evident when examining data from the CARDIOTOX-2020 registry. The registry revealed that 37.5% of chemotherapy patients experienced cardiotoxicity (with a 95% confidence interval of 34.22-40.8%). Among these, 31.6% exhibited mild damage, 2.8% experienced moderate damage, and 3.1% experienced severe damage or myocardial dysfunction. The mortality rate was exceptionally high for the severely affected group, with 22.9 deaths per 100 patients per year compared to 2.3 deaths per 100 patients per year for the other groups (95% CI 5.5-19.2, P <0.001)
Despite significant advancements in oncology care protocols, doxorubicin, an anthracycline drug in use since the 1960s, remains widely employed despite its substantial cardiotoxic potential Doxorubicin is used to treat various malignancies, including breast, colorectal, and hematological malignancies. The dose-dependent cardiotoxicity of doxorubicin manifests through diverse pathophysiological pathways, culminating in a general endpoint (HF) Despite extensive research focusing on this drug, there are still many questions regarding the pathogenesis of anthracycline cardiomyopathy.
The importance of this topic lies in the absence of consensus on establishing a cardio-oncological continuum to prevent chemotherapy-induced cardiovascular complications. Despite the existence of consensus protocols for oncological care established by authoritative medical societies, , clinical practice often reveals a significant number of non-responders to standard prevention and treatment strategies , with statistically insignificant deviations in HF incidence
A limitation of this field of study is that fundamental studies were carried out by modeling cardiotoxicity with a single chemotherapy drug in laboratory animals, such as rats and cell lines Moreover, different approaches to modeling chemotherapy-induced cardiac pathology have been employed, making it difficult to compare different datasets.
To address this issue, this article presents the author’s model of cardiomyopathy resulting from administration of the AC mode of chemotherapy, which consists of doxorubicin and cyclophosphamide. The AC mode of chemotherapy is a widely used strategy for treating various malignancies, particularly breast cancers.
This model aims to provide a comprehensive understanding of the mechanisms underlying cardiovascular complications from chemotherapy and offer insights for developing effective preventive strategies.
Study design of the author’s model of cardiomyopathy
The presented model ( Fig. 1 ) was tested in a large series of studies and was designed in strict accordance with the provisions of the Convention for the Protection of Vertebrate Animals for Experimental and Other Scientific Purposes (Strasbourg, 1986; ed. Strasbourg, 2006), international legislation on animal protection for scientific purposes (Directive 2010/63/EU) and the ARRIVE Guidelines.
This experimental model was developed only for rats and adhered to a randomized controlled experimental in vivo study design. The animals were kept in plastic cages with wood shavings at equal amounts per chamber at a temperature of 22-24°C, under 12-hour daylight conditions, and had free access to food and water. The study was comprised of two groups divided by simple randomization using the RAND function of Microsoft Excel.
The Group N1 (control) consisted of rats that were intraperitoneally injected with a single dose of 10 ml/kg sodium chloride solution three times a week for two weeks. In Group N2 (comparison 1), rats were used to study chronic cardiotoxicity by receiving intraperitoneal doses of the components of the AC mode chemotherapy. They were given a single dose of 2.5 mg kg -1 of doxorubicin hydrochloride and 25 mg kg -1 of cyclophosphamide monohydrate, three times a week for 2 weeks. The total dosage of doxorubicin hydrochloride administered was 15 mg kg -1 , and for cyclophosphamide monohydrate, it was 150 mg kg -1 over the course of the study. To investigate the cardio- and vasoprotective effects of cardioprotective compounds, groups N3 and N4 can be further designed. In group N3, alongside modeling chemotherapy-induced cardiomyopathy as in group N2, a cardioprotective (CP) substance can be administered. Group N4 (comparison 2) can be established to administer only the CP, similar to group N3, but without chemotherapy. A schematic of the proposed model is illustrated in Fig. 1 .
On days that coincided with the administration of chemotherapy drugs, cardioprotectors were administered before the intraperitoneal administration of chemotherapy. The control point of the study was on Day 14 of the experiment. Twelve hours before euthanasia, feeding of the animals was stopped and access to water remained unrestricted. Biomaterials were collected under deep anesthesia, ensuring animal welfare and adherence to ethical guidelines, and administration of the following drugs: telazol 20 mg kg -1 intramuscularly and xylazine 6 mg kg -1 intramuscularly.
The presence of cardiomyopathy was confirmed both morphologically and by echocardiographic assessment of left ventricular ejection function, along with evaluation of myocardial tissue damage and insufficiency markers (troponin I and NT pro-BNP). A schematic overview of the proposed model is shown in fig. 1 .
Pathomorphological characteristic of the heart
In the model described above, cardiomyopathy was noted during macroscopic examination, characterized by myocardial hypertrophy observed as an increase in heart mass, apex smoothing, heart chamber dilatation, and subepicardial adipose tissue proliferation. The myocardium has a flattened appearance on sectioning with foci of uneven venous congestion and cardiosclerosis. Cardiomyopathy was confirmed statistically by a significant increase in somatometric parameters. In particular, it is worth noting the changes in the heart/body weight ratio and myocardial hypertrophy index.
Microscopically, the myocardium is heteromorphic, with multiple foci of muscle fiber disintegration and hypereosinophilia of the sarcoplasm. Generally, degenerative changes are both coagulative and colliquative.
Myocardial muscle fibers were homogenized. In some places, a morphological pattern of “disarray” of cardiomyocytes was noted. Diffuse focal fragmentation and wave-like deformation of myofibrils with zones of focal myocytosis were observed. Vacuolar changes are accompanied by disruption of contractile elements, especially irregular clumps of the Z-band. Ultrastructural mitochondrial membrane disruption and swelling as well as scattered accumulation of residual bodies were observed. The affected cardiomyocytes were in a state of clumpy-like degeneration; in some places, zone foci of necrosis were visible. The nuclei of cardiomyocytes were polymorphic, with signs of chromatin margination in some areas. There are foci of inflammatory cells infiltration in the myocardial stroma. The endocardium is thickened owing to separate areas of swelling and fibrosis, and multiple small areas of subendocardial adipose tissue are noted.
The myocardial stroma is in a state of pronounced perivascular and interstitial edema with uneven plethora. The volume of adipocytes in the subepicardial and perivascular adipose tissue increased, and the thickness of adipose tissue with inflammatory infiltration were determined.
Aggregates of red blood cells were observed in intramyocardial arteries and veins. Stasis was noted in capillaries, with some micronecrotic changes in areas of the vessel wall and diapedetic hemorrhages. Some vessels are in a state of destructive-proliferative vasculitis, and in some places turn into hyalinosis and fibrosis of their walls.
On slides stained with Lie stain (also known as hematoxylin-based fuchsine-picric acid [HBFP] stain), multiple foci of fuchsinophilic myocardial lesions (fuchsinophilic substrate) were observed, indicating myocardial damage.
Fibrosis of the myocardium and blood vessels was visualized on slides stained with Malori trichrome stain.
On slides stained with alcian blue, signs of alcian degeneration and methochromasia were noted, indicating the development of mucoid swelling of the myocardial stroma associated with endocardial infiltration of glycosaminoglycans.
On slides stained with PAS, alternating foci of increased and decreased glycogen content were noted, which indicated hypoxia and metabolic disorders of cardiomyocytes.
The overview of above-mentioned microscopic changes is presented on the fig. 2 .
