Headnote
ABSTRACT
Introduction: Several studies indicate that the consumption of poliinsaturated fatty acids (PUFAs) of animal origin has protective effects on cardiovascular events, most of them showing an inverse association between high levels of these acids and the risk of cardiovascular disease (CVD).
Objective: Considering the high cost of PUFA supplementation, we aimed to analyze its cardioprotective effect, originating from low-added-value fish.
Theoretical framework: Knowing that perinatal asphyxia is the third leading cause of neonatal death in the world, it becomes even more important to carry out this work, with a view to analyzing the possible benefits of PUFAs in protecting the cardiac tissue of hypoxemiated neonatal animals.
Method: Low-cost fish samples were identified, and their fatty acid profile was determined, and used to prepare experimental diets offered in the biological trial lasting 60 days. In the experiment, mice were subjected to hypoxemic insult in the immediate postnatal period.
Results and discussion: The results demonstrate that the profile of omega 3, 6 and 9 fatty acids found in the fish used were also incorporated by the diets prepared with them. The animals subjected to hypoxia that had fish added to their diets presented higher consumption than the others, although this did not influence their weight gain, which was statistically similar among all groups. Furthermore, the hypoxia process seems to have impaired the incorporation of PUFAs, given the changes observed in the macro and microscopy of the cardiac tissue. Furthermore, when comparing the fish species analyzed, the dourado species seems to qualitatively influence the cardiac morphological pattern when compared to the red snapper species.
Implications of the research: This study is of interest to several areas. It begins with the identification of fishery resources and fish of low commercial value but with good nutritional quality that can be encouraged to increase their consumption by the Brazilian population, whose diet is scarce in quality fish. This is due not only to the high cost that fish consumption currently represents for the population, who opt to consume protein sources of low nutritional value such as sausages and nuggets, which, although they have low nutritional value, are also low cost when compared to fish, unfortunately being an attraction. Furthermore, the discussion about the importance of these essential fatty acids as protectors against cardiovascular events is quite useful, with the aim of promoting accessible strategies to increase their consumption, and this project could even serve as a subsidy for, in the future, thinking about strategies for enriching AGEs at a national level in industrialized products, as is already done today with Folic Acid and Iron in flour and Iodine in table salt, to prevent nutritional deficiencies.
Originality/value: Thus, it is expected that in the future, based on the development of by-products based on the fish samples studied in this project, they will be included in children's diets through school meals in public schools, since these must follow adequate nutritional criteria, as provided for in the National School Feeding Program (PNAE).
Keywords: Polyunsaturated Fatty Acids, Heart, Fish, Cardioprotection, Postnatal Hypoxia, Supplementation.
RESUMO
Introducáo: Diversos estudos apontam que o consumo de ácidos graxos poli-insaturados (PUFAs) de origem animal exerce efeitos protetores sobre eventos cardiovasculares, a maior parte deles mostrando uma associação inversa entre altos niveis destes ácidos e riscos de doenca cardiovascular (DCV).
Objetivo: Tendo em vista o custo elevado da suplementação dos PUFAs, objetivamos analisar seu efeito cardioprotetor, oriundo de pescado de baixo valor agregado.
Referencial teórico: Sabendo que a asfixia perinatal é a terceira causa de morte neonatal no mundo, torna-se ainda mais importante a realização deste trabalho, com vistas a analisar os possíveis beneficios dos PUFAs na proteção do tecido cardíaco de animais neonatos hipoxemiados.
Método: Foram identificadas amostras de pescado de baixo custo, que tiveram seu perfil de ácidos graxos determinado, sendo utilizadas para confecção de rações experimentais ofertadas no ensaio biológico com duração de 60 dias. No experimento, camundongos foram submetidos a insulto hipoxémico no pós-natal imediato.
Resultados e discussáo: Os resultados demonstram que o perfil de ácidos graxos ómega 3, 6 e 9 encontrados no pescado utilizado também foram incorporados pelas rações confeccionadas com eles. Os animais submetidos a hipóxia que tiveram pescado acrescido as suas rações apresentaram consumo superior aos demais, embora isso náo tenha influenciado no seu ganho de peso, estatisticamente semelhante entre todos os grupos. Ademais, o processo da hipóxia parece ter prejudicado a incorporação dos PUFAs, visto as alterações observadas na macro e microscopia do tecido cardíaco. Além disso, ао compararmos as espécies de pescado analisadas, a espécie dourado parece influenciar qualitativamente o padráo morfológico cardíaco quando comparado a espécie pargo.
Implicações da pesquisa: Esse estudo contém interesses para varias áreas. Começando pela identificação de recursos pesqueiros e pescado de baixo valor comercial, mas como boa qualidade nutritiva que possam ser estimulados ao aumento de seu consumo pela população brasileira, que possui uma alimentação escassa de pescado de qualidade. Tal fato deve - se náo só ao elevado custo que, atualmente, o consumo de peixe representa para a populacáo, que opta por consumir fontes proteicas de baixo valor nutritivo como embutidos, tais como salsichas e nuggets, que, embora tenha baixo valor nutritivo, apresentam também baixo custo quando comparadas ao pescado, sendo um atrativo, infelizmente. Além disso, a discussáo acerca da importáncia destes ácidos graxos essenciais como protetores sobre eventos cardiovasculares é bastante proveitosa, no intuito de que se promovam estratégias acessíveis para aumento do consumo destes, podendo servir esse projeto, inclusive, como subsídio para, no futuro sejam pensadas estratégias de enriquecimento de AGEs a nivel nacional de produtos industrializados, como hoje já se faz com o Ácido Fólico e Ferro em farinhas e о lodo no sal de cozinha, para prevenção de carências nutricionais.
Originalidade/valor: Dessa forma, espera - se no futuro, a partir do desenvolvimento de subprodutos a base das amostras de pescado estudadas neste projeto, incluir os mesmos na alimentação infantil através da merenda escolar de escolas publicas, uma vez que estas devem seguir critérios nutricionais adequados, como previsto no Programa Nacional de Alimentação Escolar (PNAE).
Palavras-chave: Ácidos Graxos Poli-insaturados, Coração, Pescado, Cardioproteçäo, Hipóxia Pós-Natal, Suplementação.
RESUMEN
Introducción: Diversos estudios indican que el consumo de ácidos grasos poliinsaturados (AGPI) de origen animal tiene efectos protectores sobre eventos cardiovasculares, mostrando la mayoría de ellos una asociación inversa entre niveles elevados de estos ácidos y el riesgo de enfermedad cardiovascular (ECV).
Objetivo: Considerando el elevado coste de la suplementación con PUFA, nos propusimos analizar su efecto cardioprotector, proveniente de pescado de bajo valor añadido.
Marco teórico: Conociendo que la asfixia perinatal es la tercera causa de muerte neonatal en el mundo, cobra aún mayor importancia la realización de este trabajo, con el fin de analizar los posibles beneficios de los PUFAs en la protección del tejido cardíaco de animales neonatales hipoxemiados.
Método: Se identificaron muestras de peces de bajo costo y se determinó su perfil de ácidos grasos, que se utilizaron para preparar alimentos experimentales ofrecidos en el ensayo biológico con una duración de 60 días. En el experimento, los ratones fueron sometidos a un ataque hipoxémico en el período postnatal inmediato.
Resultados y discusión: Los resultados demuestran que el perfil de ácidos grasos omega 3, 6 y 9 encontrados en los pescados utilizados fueron también incorporados en las raciones elaboradas con ellos. Los animales sometidos a hipoxia a los que se añadió pescado a su dieta mostraron un mayor consumo que los demás, aunque esto no influyó en su ganancia de peso, que fue estadísticamente similar entre todos los grupos. Además, el proceso de hipoxia parece haber perjudicado la incorporación de PUFAs, dados los cambios observados en la macro y microscopía del tejido cardíaco. Además, al comparar las especies de peces analizadas, la especie dorado parece influir cualitativamente en el patrón morfológico cardíaco en comparación con la especie pargo.
Implicaciones de la investigación: Este estudio tiene interés para varias áreas. A partir de la identificación de recursos pesqueros y peces de bajo valor comercial, pero con buena calidad nutricional que puedan ser incentivados a aumentar su consumo por la población brasileña, que tiene una dieta escasa en pescado de calidad. Este hecho se debe no solo al alto costo que actualmente representa el consumo de pescado para la población, quienes optan por consumir fuentes de proteína con bajo valor nutricional como salchichas y nuggets, que si bien tienen bajo valor nutricional, también tienen un bajo costo si se comparan con el pescado, siendo lamentablemente un atractivo. Además, la discusión sobre la importancia de estos ácidos grasos esenciales como protectores contra eventos cardiovasculares es bastante útil, con el objetivo de promover estrategias accesibles para incrementar su consumo, e incluso este proyecto podría servir como subsidio para, a futuro, pensar en estrategias de enriquecimiento de AGEs a nivel nacional en productos industrializados, como ya se hace hoy con el Ácido Fólico y el Hierro en las harinas y el Yodo en la sal de mesa, para prevenir deficiencias nutricionales.
Originalidad/valor: De esta forma, se espera que en el futuro, a partir del desarrollo de subproductos a partir de las muestras de pescado estudiadas en este proyecto, los mismos sean incluidos en las dietas infantiles a través de las comidas escolares de las escuelas públicas, ya que estas deben seguir criterios nutricionales adecuados, tal y como prevé el Programa Nacional de Alimentación Escolar (PNAE).
Palabras clave: Ácidos Grasos Poliinsaturados, Corazón, Pescado, Cardioprotección, Hipoxia Postnatal, Suplementación.
1 INTRODUCTION
The heart is a pulsatile pump composed of two atria and two ventricles. The ventricles provide the main pumping force that propels blood through the pulmonary circulation from the right ventricle or the peripheral circulation from the left ventricle. Special mechanisms of the heart promote the continuous succession of cardiac contractions, called cardiac rhythm, by transmitting action potentials through the cardiac muscle causing the rhythmic beating of the heart (Guyton & Hall, 12th edition). Cardiovascular diseases (CVD) are a series of diseases involving the circulatory system (heart and blood vessels), including atherosclerosis, coronary heart disease, acute myocardial infarction, and heart failure (Loscalzo et al ., 2024). According to the latest statistics, 19.05 million people worldwide died from CVD in 2020, an increase of 18.71% compared to 2010, which is more than the total number of deaths caused by cancer and chronic respiratory diseases (Oliveira et al ., 2024).
Cardiac ischemia is a consequence of an imbalance between the supply and demand of oxygen by the myocardium, which leads to significant structural changes. In this sense, it 1s observed that ischemia induces a decrease in cellular glycogen, relaxation of myofibrils and rupture of the sarcolemma, which can culminate in generalized edema. In addition, if the ischemia is not resolved, cellular necrosis may occur which, combined with the attempted repair, results in the formation of scar tissue in the myocardial tissue (Emrich et al ., 2021, Del Buono et al ., 2022, Bradley & Berry, 2022). Perinatal asphyxia occurs when the baby is deprived of oxygen close to birth, and can occur before, during or immediately after birth (WHO, 2022). It can be defined as an injury suffered by the fetus or newborn due to poor oxygenation (hypoxia) and/or poor perfusion (ischemia) of multiple organs.
Regarding the cardiovascular system, transient myocardial ischemia is common, with signs of predominantly right-sided heart failure and tricuspid insufficiency (regurgitation murmur). Oxygen deprivation can also generate cardiogenic shock resulting from global myocardial ischemia or necrosis and rupture of the papillary muscles. Clinically, we see signs of shock with weak pulses, muffled heart sounds, poor capillary filling, difficulty in venous access, tachycardia or bradycardia (Margotto, 2021).
Omega-3 (n-3) fatty acids are mainly derived from fish and fish oils and are protective against atherosclerotic heart disease, stroke and sudden coronary death. In addition, they are potentially therapeutic due to their immunomodulatory and anti-inflammatory effects. N-3 has several beneficial effects on health, such as improved blood pressure, cardiac function, endothelial and vascular function, arterial compliance, lipoprotein and lipid metabolism, and reduced formation of cytokines, monocytes and neutrophils. The n-3 fatty acids that have the greatest potency for these specific actions are the long-chain polyunsaturated acids of marine origin, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Mori; Beilin, 2004).
Along with omega 3, omega 6 has also been portrayed in the literature as beneficial for health in the prevention and/or treatment of diabetes, cancer, inflammation, cardiovascular diseases and neurological diseases. Several studies also point to omega 9 as excellent for cardiovascular health, neurodegenerative diseases and anti-inflammatory effects; however, there is controversy regarding the adverse effects associated with its consumption (Wang, et al., 2024).
Among the nutritional sources of these fatty acids, fish stands out, which differs from other foods due to its excellent bioavailability of vitamins and minerals. In addition, fish has a high quantity of long-chain polyunsaturated fatty acids that are highly important for health due to their antithrombotic activity. Finally, its proteins contain all the essential amino acids for human nutrition (Sartori; Amancio, 2012). Due to the importance of omega-3, it is recommended to consume fish or other seafood once or twice a week, especially fatty fish (dark meat) that are richer in DHA and EPA; this consumption is associated with health benefits such as reduced risk of stroke, Alzheimer's disease, depression and deaths from heart problems. The Food and Drugs Administration (FDA) recommends the use of approximately 2 grams/day of this fatty acid. The American Health Association (AHA) recommends the use of 1 gram daily for people with heart disease and 2 to 4 grams for people seeking to reduce triglycerides, both under professional supervision (HARVARD, 2012).
In this context, it appears that the intake of these polyunsaturated fatty acids in the diet is an important factor in reducing the risk of cardiovascular diseases, and this function is even recognized by ANVISA (National Health Surveillance Agency), whose functional claim highlights that the consumption of omega-3 fatty acids helps maintain healthy triglyceride levels, as long as it is associated with a balanced diet and healthy lifestyle habits (ANVISA, 2019). However, there is still a need for studies that evaluate the preventive potential of this fatty acid, specifically related to damage to cardiac tissue. Likewise, little has been previously studied about the best sources of lower-cost marine fish that can serve as a source of these fatty acids and, thus, participate in this therapeutic effect.
Therefore, this study seeks to evaluate the possible benefits of omega-3, 6 and 9 in cardiac tissue subjected to hypoxia as well as the functionality of these acids from low-cost fish species.
2 METHODOLOGY
2.1 SELECTION OF SPECIES
Initially, through visits to the Municipal Fish Market of Macaé/RJ and interviews with local traders, the most commonly sold fish species were identified, as well as those with the lowest cost, and two were selected for use in this study. The selection and collection of species was carried out with the help of veterinarians from the Fundacáo Instituto da Pesca do Estado do Rio de Janeiro (FIPERD).
2.2 DETERMINATION OF THE FATTY ACID PROFILE OF FISH SAMPLES
To analyze the fatty acid profile in the fish samples, lipid extraction, saponification and methylation of fatty acids were performed (Lenzi et al., 2011), which was done in duplicate in the two identified samples.
2.3 BIOLOGICAL ASSAY
All experimental procedures were performed in accordance with the standards established in the conventional guide for animal experimentation (NIH Publication No. 85-23, revised in 1996) and the national recommendations imposed by the Arouca Law (11.794/2008). The work was submitted and approved by the Animal Research Ethics Committee of the CCS/UFRJ for certification purposes under number CEUA/CCS 036/22.
For the biological assay, the two species with the highest omega-3 content and lowest cost among those analyzed were chosen. In addition, species with a significant concentration of the fatty acid DHA were chosen, due to its antioxidant effect. Thus, samples of Dourado (Coryphaena hippurus) and Red Snapper (Pagrus major) were the fish identified with the highest content of AGEs (essential fatty acids) of lowest economic value that were used to prepare the experimental diets offered to the animals, as described below.
In the biological assay, 30 Swiss mice (Mus musculus) were used, 24 females and 6 males, from the colony of the Animal Experimentation Bioterium of the Division of Research in Bioactive Products and Biosciences of the CM UFRJ-Macaé, which were polygamously mated in the proportion of 1 male / 4 females. The pregnant females were separated into three groups (n = 3) different in terms of the composition of the food consumed: Control Group (CG) receiving casein-based food, Golden Group (GD) receiving casein-based food and golden fish, and Red Snapper Group (GP) receiving casein-based food and red snapper fish. The offspring of these animals originated the experimental groups themselves, as will be outlined below.
The rations were prepared in accordance with AIN-93, and it is important to highlight that the lower casein content in the dorado and snapper rations is due to the protein content contained in these species.
2.4 EXPERIMENTAL DESIGN
On the first day of birth (РО), half of the pups assigned to each dietary group went through the hypoxia process, thus creating 6 groups in total:
* Control group (CG, n=8): receiving casein-based feed;
* Hypoxic control group (HCG, n=8): receiving casein-based feed, having suffered hypoxia;
* Golden control group (GDC, n=8): receiving casein-based feed added with freeze-dried golden flour;
* Hypoxic golden group (GDH, n=8): receiving casein-based feed added with freeze-dried golden flour, having suffered hypoxia;
* Red snapper control group (CPG, n=8): receiving casein-based feed added with freeze-dried red snapper meal and
* Hypoxic red snapper group (РНС, n=8): receiving casein-based feed added with freeze-dried red snapper flour, having suffered hypoxia.
To perform hypoxia on the animals, within two hours after birth, the pups destined for hypoxemic insult were separated from their mothers for 20 minutes to acclimatize and placed in a special chamber, hermetically sealed for 60 seconds. After this time, they were placed back with their respective mothers. The animals that did not undergo this process were separated from their mothers for 20 minutes to ensure similar treatment to the others.
The animals were kept in collective cages (n=3/cage), with constant temperature (24 + 2°C) and controlled lighting, 12/12h light-dark cycle, receiving water and food ad libitum throughout the trial. Throughout the experiment, to determine the Feeding Profile, the animals were weighed twice a week. The food offered was weighed on alternate days to verify the amount ingested by each animal. The food offered and the remaining food of the animals were counted.
At the end of 60 days of experimentation, the animals were anesthetized and euthanized by decapitation for collection of hearts, which were weighed and evaluated macroscopically, according to the following criteria: shape, volume or size, color, presence of lesions and any other apparent alteration among the experimental groups when compared to GC and GCH. For histopathology, the heart samples were placed in cassettes, stored in 10% formaldehyde with pH between 0.6 and 0.7 for 48 hours, and submitted to routine techniques for preparing slides. The sections were cut with 3.0 um thickness for mounting the slides, which were stained with hematoxylin eosin. After this process, they were observed under an optical microscope (model Novel BM2100) and photographed using a Tucsen camera, model ISH300, 3.0MP, through the TCapture 5.1.1 program for histopathological description.
2.5 STATISTICAL ANALYSIS
Data were analyzed using GraphPad Prism, version 5.0, and expressed as mean + standard error of the mean. The Shapiro-Wilk test was performed to assess data normality. Statistically significant differences between groups were calculated by applying a One-Way Analysis of Variance (ANOVA) followed by the Tukey multiple comparisons test. The T-test with Mann Whitney post-test was used to test the results of the hypothesis of non-normal distribution. Differences between the experimental groups and the control group were considered statistically significant when p<0.05.
3 RESULTS AND DISCUSSIONS
In the analysis of results, we sought to evaluate the influence of the consumption of rations with added fish on the development, growth, as well as the cardiac architecture of hypoxic or non-hypoxic animals.
The data expressed in Figure 2 refer to the findings of the chromatographic analysis performed on the fish samples used in the biological assay. A higher concentration of the fatty acids EPA (0.01) and DHA (0.13) was observed in the Pink Snapper ( Pagrus major ), also observed in the concentrations of omega 3 (0.14), omega 6 (0.08) and omega 9 (0.21); when compared to the Golden Fish ( Coryphaena hippurus), where: EPA=0.00; DHA=0.09; omega 3=0.09; omega 6=0.06 and omega 9=0.16.
Regarding the data related to the total feed consumption of the animals at the end of 60 days of experiment (Figure 3), these presented statistical significance. Observing the control groups (Which did not suffer hypoxia), the GDC and GPC were equal and are statistically superior to the GC (p<0.0001). Regarding the hypoxemia groups, they were different from each other. GCH and GDH were equal and are inferior to the GPH, which presented a higher, statistically significant value (p=0.0248). Some studies carried out in fish species indicate that intermittent or chronic hypoxia can cause reduced feed consumption, reduced growth, increased diseases and mortality, and its effects are still poorly understood (Remen, et al . 2013). However, in our experiment, the hypoxemic process did not present a significant influence on the consumption and weight gain of the mice.
Fish, regardless of its commercial value, is an easily digestible food with a satisfactory content of proteins, unsaturated fats, vitamins and minerals, compared to other products of animal origin. From a nutritional point of view, they have good sensory and nutritional characteristics: tasty meat, low fat and calorie content. It has a protein content comparable to beef, pork and poultry. In addition, fish has been identified as a food with a high content of AGEs, omega-3 (EPA and DHA); in addition to omega 6 and omega 9. The consumption of these lipids is associated with numerous functions, such as reducing the risk of cardiovascular diseases, maintaining adequate triglyceride levels and aiding in the wound healing process, as highlighted by Grandini et a/., (2020).
Figure 4 presents the data regarding the assessment of the consumption of omega 3, 6 and 9 of the groups of mice, according to their food consumption and the composition of the specific food that was given to each group. It is important to highlight that to calculate the consumption of omega 3 and 6, the content of these fatty acids present in the soybean oil used in the preparation of the food was also considered, according to the nutritional information on the label (SOYA, 2024).
Animal sources of omega-3 are mainly fish and marine products. EPA and DHA are found in fish of the Scombridae, Clupeidae and Salmonidae families. Alpha-linolenic acid (ALA) in animals that have desaturase enzymes is an initial precursor for the formation of EPA and DHA. All fish have the alpha 6-desaturase enzyme, an enzyme that acts in the initial desaturation of ALA, while alpha 5-desaturase, necessary for the formation of EPA, is present only in diadromous fish (fish that migrate between fresh and salt waters). The exclusive intake of vegetable ALA is insufficient for the demand of the human body, so it is necessary to consume animal sources of omega-3 (Souto, et al.; 2022). However, algae oils are plant sources and have great potential for increasing the plasma concentration of EPA and DHA in the body, but their functional action requires further studies (Viana, 2022).
Figures 5 and 6 show the data on the animals' weight gain throughout the experiment, as Well as the data on the relative weight of the hearts of these animals. Although the feed consumption data were statistically different between the groups, this did not influence the body weight gain, which was statistically similar. Even so, a significant numerical difference can be observed between the groups, specifically in those that received fish added to their diets, which is suggested to be related to the nutritional quality of the fish. There was no statistical difference between the relative heart weights of the experimental groups.
Analyzing the cardiac macroscopy of the animals, it is noted that the hearts subjected to the hypoxic insult presented loss of color, possibly attributed to oxygen deprivation. This becomes more evident in the hypoxemia control group (HCG), when compared to the other hypoxemia groups (РНС and DHG), since they did not receive any of the diets supplemented with fatty acids with cardioprotective effects, as already described in the literature (Maral & Yashwant, 2024; Jin & Makoto, 2016; Alagawany et al, 2022). In histopathology, we observed that the groups that suffered hypoxia presented greater disorganization of the cardiac fibers, as well as nuclear pleomorphisms, evidencing tissue damage. However, comparing only the groups of animals subjected to hypoxia, we noticed that the animals that received the diet with added Pargo fish (GPH) demonstrated better macro and microscopic appearance, when compared to the hypoxic control group (GCH) and the group that consumed diet based on Dourado fish (GDH). These findings corroborate those observed in Table 3, where we observed that the estimate for the estimated total consumption (g) of omega 3, omega 6, omega 9 fatty acids, for each experimental group, in 60 days of duration of the experiment, presented values of higher consumption for the animals of GPH.
Finally, comparing the control groups (not subjected to hypoxia: GC, GPC, GDC), we noticed that the animals that consumed feed with added Pargo fish (GPC), showed a qualitatively superior aspect in the histopathology, with cardiac muscle cells in an elongated and branched shape, with one or two nuclei, perfectly organized.
With these findings, we can infer that the content of omega 3, omega 6 and omega 9 fatty acids, found in greater quantities in the snapper fish, consumed by mothers and offspring throughout the experiment, positively influenced cardiac development, as well as appearing to have attenuated the impact caused by the hypoxic insult at the birth of the animals.
4 CONCLUSION
We conclude that further studies are needed to demonstrate the possible benefits and protective factors of omega-3, -6 and -9 fatty acids after hypoxemic lesions in myocardial tissue. Although there are already numerous studies in the literature that demonstrate the advantages of PUFA supplementation, both in the nervous system and in the cardiovascular system, the objective of the present study was to analyze the efficacy of the consumption of these fatty acids in the daily diet and not as a supplement. Thus, it was observed that the hypoxia process seems to have impaired the incorporation of PUFAs, given the changes observed in the macro and microscopy of the cardiac tissue, but further studies are needed to corroborate what was described in this experiment.
ACKNOWLEDGMENTS
We would like to thank the Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro and the Deputy Secretary of Higher Education of the City of Macaé for granting funding for this research, respectively, in the notices "7th Edition of the Research Program for the SUS: shared management in health (PPSUS) and Macaé Conecta 2021 and 2022" and also for granting Scientific Initiation scholarships to students participating in this project. We would also like to thank PIBIC/UFRJ and PIBIC/CNPq for granting Scientific Initiation scholarships.
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