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Ultrastructural effects of diabetes in the right atrium cardiomyocytes of elderly Wistar rats
Journal Pre-proof Ultrastructural effects of diabetes in the right atrium cardiomyocytes of elderly Wistar rats Natalie Souza de Andrade, Kemily Loren Barros Chucata, Walkyria Villegas Magalhães, Ricardo Aparecido Baptista Nucci, Nicolas Da Costa-Santos, Igor Roberto Dias, Hunter Douglas de Souza Lima, Laura Beatriz Mesiano Maifrino, Romeu Rodrigues de Souza PII:
S1054-8807(19)30346-1
DOI:
https://doi.org/10.1016/j.carpath.2019.107181
Reference:
CVP 107181
To appear in:
Cardiovascular Pathology
Received Date: 23 September 2019 Revised Date:
10 October 2019
Accepted Date: 11 November 2019
Please cite this article as: Souza de Andrade N, Barros Chucata KL, Magalhães WV, Baptista Nucci RA, Da Costa-Santos N, Dias IR, Douglas de Souza Lima H, Mesiano Maifrino LB, Rodrigues de Souza R, Ultrastructural effects of diabetes in the right atrium cardiomyocytes of elderly Wistar rats, Cardiovascular Pathology, https://doi.org/10.1016/j.carpath.2019.107181. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Inc. All rights reserved.
Ultrastructural effects of diabetes in the right atrium cardiomyocytes of elderly Wistar rats Natalie Souza de Andrade1,2, Kemily Loren Barros Chucata1, Walkyria Villegas Magalhães1, Ricardo Aparecido Baptista Nucci1,3,*, Nicolas Da Costa-Santos1, Igor Roberto Dias1, Hunter Douglas de Souza Lima1, Laura Beatriz Mesiano Maifrino1,4, Romeu Rodrigues de Souza1,5 1
Department
of
Physical
Education,
Laboratory
of
Morphological
and
Immunohistochemical Studies, São Judas Tadeu University, São Paulo, SP, Brazil 2
Uninove University, São Paulo, SP, Brazil
3
Department of Pathology, University of São Paulo Medical School, São Paulo,
SP, Brazil 4
Dante Pazzanese Institute of Cardiology, São Paulo, SP, Brazil
5
Department of Anatomy, Institute of Biomedical Sciences of University of São
Paulo, São Paulo, SP, Brazil
Corresponding author at: São Judas Tadeu University and University of São Paulo Medical School, São Paulo, Brazil. E-mail address: [email protected] (R.A.B. Nucci)
ABSTRACT The present study aimed to evaluate the effects of diabetes on quantitative parameters of right atrial cardiomyocytes of elderly rats. Wistar rats (14-mo of age) were divided into two groups: streptozotocin-diabetic rats (DG); and control rats (CG). The groups were sacrificed at 16 months. Ultrafine sections of the right atrium were analyzed by electron microscopy. In elderly diabetic animals, histograms of the frequency distribution of natriuretic peptides according to their size showed increased number of small and medium peptides in relation to large peptides, which increased its numerical density leading to a decrease in the mean diameter of both natriuretic peptides. However, elderly diabetic animals remained normotensive. No significant difference was observed between the groups for the volume density of mitochondria, endoplasmic reticulum and Golgi apparatus. In conclusion, elderly diabetic rats showed increased functional activity of atrial cardiomyocytes with greater production of natriuretic peptides in association with a quantitative maintenance of cytoplasmic components. Key words: Aging; Atrial cardiomyocytes; Electron microscopy; Morphometry; Streptozotocin.
1. Introduction Diabetes mellitus (DM) have direct effects on cardiac muscle, and its effects on atrial cardiomyocytes have been studied in experimental models, e.g., rats [1-5]. Atrial cardiomyocytes produce atrial natriuretic peptide (ANP) and B-type or brain natriuretic peptide (BNP) that act to control blood pressure [6-8]. Although, the effects of DM on the production of ANP and BNP in atrial cardiomyocytes have been studied in young animals demonstrating an important relationship between cardiomyocyte function and these peptides [914], there is still a lack of studies with elderly diabetic rats [15]. Additionally, it is suggested that the production of both ANP and BNP in cardiomyocytes is directly related to the function of mitochondria, Golgi complex and endoplasmic reticulum, which, in turn, has an influence on the volume densities of these organelles [9,10,16]. However, the effects of DM on these components in elderly rats have not been elucidate. The present study aimed to evaluate the effects of diabetes on quantitative parameters of right atrial cardiomyocytes of elderly rats through morphometric techniques on electronic photomicrographs [17,18]. 2. Methods All experimental procedures conformed to the guiding principles of the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996. The Institutional Animal Care and Use Committee of the university approved the experimental protocol (protocol number 100/08). The animals used in the present study were male Wistar rats (14-month-old) purchased from the Laboratory Animal Center of Institute of Biomedical Sciences of University of São Paulo, São Paulo, Brazil. Animals were divided in two groups (n = 8/per group): control group (CG); and rats submitted to diabetes induction (DG). The animals were housed in plastic cages with access to food and water ad libitum and maintained on a 12 hours light/dark cycle at room temperature (23–26°C). The food stuff consumption of each rat per day and night was measured regularly.
2.1. Induction of DM DM was induced in the animals at age of 14 months by an intravenous injection of Streptozotocin (70 mg / kg, Sigma) after one overnight fast (8-10 hours) [4]. The control group received at age of 14 months only vehicle (10 mm citrate buffer, pH 4.5) after an overnight fast. After 30 days of induction to DM, a blood sample of the animals from the DG was collected and sent to the laboratory, and DM-compatible glycemic levels were found. 2.2. Euthanasia and sample collection At the end of the experiment (16 months age), the systolic pressure was obtained indirectly through the tail cuff method [19], and the animals had their final glycemia evaluated by glycosometer to comparison. Animals from both groups were euthanized with sodium pentobarbital and their ventricles perfused with heparinized saline solution (1 mg / 300 ml solution). The right atria were removed and reduced to fragments of approximately 4 mm³. The fragments were placed in glutaraldehyde 2% in sodium cacodylate buffer solution (0.2 m, pH 7.3) for three hours. The fragments were then washed three times with the same buffer solution for 5 minutes at a time, and subsequently placed in a solution of 1% osmium tetroxide in sodium buffer and cacodylate for 2 hours. The fragments remained overnight in 0.5% uranyl acetate with sucrose (54 mg / 100 ml) and after washing with the buffer were dehydrated in the growing series and absolute alcohol and propylene oxide for 8 hours under rotation. The fragments were then included in pure resin for 5 hours and finally left in the same resin at 60°C for 5 days. The ultr afine sections were obtained with a diamond knife in an ultramicrotome (Sorvall MT-2), and after having been contrasted with uranyl acetate and lead citrate were used to perform cardiomyocyte photomicrographs in the transmission electron microscope (Jeol) of the Institute of Biomedical Sciences of University of São Paulo. 2.3. Morphometric evaluation Electron micrographs of ten areas per animal, i.e., a total of 160 areas, sampled by random [20], were taken with an increase of ×15000. We used a quadrangular area of 54 µm2 for counting ANP and BNP numerical density in the cardiomyocyte. Using a Zeiss microscope, coupled to a computerized image analysis program (Axio Vision, Zeiss) from the Laboratory of Morphological and
Immunohistochemical Studies of the São Judas Tadeu University, we measured both ANP and BNP granule diameters. The largest and smallest diameter of each granule were measured, and the arithmetic averages of both measurements were used for the final calculations. With the aid of ImageJ software, we analyzed the volume density (%) of mitochondria, endoplasmic reticulum and Golgi apparatus in the cytoplasm (Fig. 1). The volume density corresponds to the area occupied by the structure in the field in percentage [17]. It was obtained using a test system equipped with 80 points (considered as 100%) placed on each photomicrograph. The following formula was used to obtain the volume density of the cardiomyocyte components: Vv [structure] = ΣP [structure] 100 / TP; where, Vv [structure] = Volume density of the structure, ΣP [structure] = number of points on the structure in question, and TP = Total number of points (80) of the test system [17,18]. 2.5. Statistical analysis Data were expressed as mean ± standard error (SEM). We conducted unpaired Student’s t-test to examine whether the groups were different regarding the variables. The statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Prism, Inc., San Diego, CA). The alpha level was set at the 0.05 level, and all tests were two-tailed. 3. Results 3.1. Values of systolic blood pressure and blood glucose Table 1 shows the final measurement of systolic blood pressure and glycemia of the animals in both groups before euthanasia. We verified that there was no significant difference between the arterial pressure values between the groups (p>0.05). However, the glycemia values in the DG animals are compatible with the presence of diabetes (p<0.05). 3.2. Morphometry The number and diameter of the granules in the right atrium cardiomyocytes in both groups of rats are shown in Fig. 2. We observed that the values for the number of granules in DG are significantly higher than CG for both types of granules (ANP and BNP) (p<0.001). On the other hand, the
diameter of the granules of ANP and BNP were significantly lower in DG when compared to CG (p<0.001). The results obtained for the distribution of frequencies (%) of the granules of ANP and BNP according to their diameter in the groups CG and DG can be observed (Fig. 2). The results for ANP show that diabetes promoted a decrease in the percentage of medium and large granules, and an increase in the percentage of small ones. In addition, the results for the BNP shows that there was a decrease in the percentage of small granules and an increase in the percentages of the medium and large granules in diabetic animals. However, Fig. 3 shows a not significant decrease (p>0.05) in the volume density of mitochondria, endoplasmic reticulum and Golgi complex in the diabetic animals when compared to control. Qualitatively, we observed preserved morphological aspects for these organelles. 4. Discussion There are two main findings in this work. First, the numerical density of ANP and BNP in cardiomyocytes was significantly higher in the diabetic elderly group compared to the control group, and the ANP and BNP granule size was significantly lower in the diabetic group than in the control group, indicating that diabetes had an influence in the production of natriuretic peptides. These results are similar to previous studies on experimental induction of DM in young animals and in diabetic patients [21-23]. Second, despite previous studies had demonstrate lesions on cytoplasmic organelles [24,25], we observed in elderly streptozotocin-diabetic rats a nonsignificant influence of this disease on the density of cardiomyocytes organelles. Additionally, these organelles were wellpreserved in both groups. According to previous works, during aging there is an increase of natriuretic peptides [26,27]. In our study, we observed that diabetes increased the numerical density of natriuretic peptides when compared to aged nondiabetic animals. The increased density of ANP and BNP granules observed in the cytoplasm of cardiomyocytes from diabetic animals, may be due to an increase in peptide production or a decrease in their secretion into the bloodstream. To corroborate with our results, a previous study in mice showed
that the secretion of these peptides was reduced in diabetic animals [21], suggesting that in diabetes, polyuria occurs with glycosuria due to hyperglycemia. It is possible that diabetic cardiomyocytes retain ANP and BNP in cardiomyocytes cytoplasm to avoid excessive loss of sodium, as these peptides, when released into the bloodstream, act in the kidneys increasing the output of sodium and water [6,16,28]. Thus, in animals with diabetes, the reduction of ANP and BNP secretion may occur to maintain extracellular sodium rates. Regarding the diameter, diabetic animals had smaller granules than those of the control animals. Furthermore, DM increased smaller ANP and medium BNP granules, which explains the decrease in mean diameter observed in the granules of diabetic animals. These results suggest that DM influenced not only the size but also the proportion of the granules in terms of their size and future studies are need to elucidate the mechanisms related to this phenomenon. We observed that diabetic animals did not show presence of arterial hypertension, which suggests that they remained normotensive. Thus, the increase of ANP and BNP production by cardiomyocytes was not due to the presence of hypertension, as both peptides have an important role in maintaining blood pressure [6], but exclusively to diabetes. Mitochondrial lesions were observed in diabetic rats indicating failure of mitochondrial control mechanisms [25]. Studies have related mitochondrial function to the balance between energy demand and nutrient supply, suggesting that changes in mitochondria may act as a mechanism for bioenergetic adaptation during pathological cardiac remodeling [25,29]. Additionally, Kordowiak et al. [24] observed a reduction of Golgi complex membranes and endoplasmic reticulum in hepatic cells of diabetic rats. In contrast, our study showed that the mitochondria, Golgi apparatus and endoplasmic reticulum of cardiomyocytes did not undergo quantitative changes in the prevalence of DM, however, our results may be related to the period of diabetes (2 months) that the animals underwent. However, our results should be examined considering the study limitations. Although, cardiomyocytes organelles were well-preserved, we did not analyzed the activity of these organelles [25]. On the other hand, future
studies should consider the period of diabetes for a better understanding of our results, as previous studies with streptozotocin-diabetic experimental models had a period above 2 months [1,4]. Although our study has limitations, we also have some advantages, as we presented ultrastructural effects of diabetes in the right atrium using standardized morphometrical techniques [17,18]. Additionally, our diabetic experimental model was in accordance with previous described in the literature [1-4]. In conclusion, elderly diabetic rats showed increased functional activity of atrial cardiomyocytes with greater production of ANP and BNP granules in association with a quantitative maintenance of cytoplasmic components. Accordingly, further studies are needed regarding the physiological and molecular mechanisms involving cardiomyocytes injury and diabetes for a better understanding of our results. Declaration of interest The authors declare not competing or financial interest. References [1] Hussein AEAM, Omar NM, Sakr H, Elsamanoudy AZ, Shaheen D. Modulation of metabolic and cardiac dysfunctions by insulin sensitizers and angiotensin receptor blocker in rat model of type 2 diabetes mellitus. Can J Physiol Pharmacol 2011;89(3):216-226. https://doi.org/10.1139/Y11-012 [2] Kato T, Yamashita T, Sekiguchi A, et al. Angiotensin II type 1 receptor blocker attenuates diabetes-induced atrial structural remodeling. J Cardiol 2011;58(2):131-136. https://doi.org/10.1016/j.jjcc.2011.06.003 [3] Musial DC, de Magalhães Galvão K, Miranda-Ferreira R, et al. Alteration of purinergic neurotransmission in isolated atria of streptozotocin-induced diabetic rats.
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[5] Maria Z, Campolo AR, Lacombe VA. Diabetes alters the expression and translocation of the insulin-sensitive glucose transporters 4 and 8 in the atria. PLoS
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[24] Kordowiak AM, Dudek B, Gryboś R. Influence of sodium bis (oxalato) oxovanadate (IV) on phospholipids in liver Golgi fractions from control and streptozotocin-diabetic rats. Comp Biochem Physiol C Toxicol Pharmacol 2000;125(1):11-16. [25] Liang Q, Kobayashi S. Mitochondrial quality control in the diabetic heart. J Mol Cell Cardiol 2016;95:57-69. https://doi.org/10.1016/j.yjmcc.2015.12.025 [26] Korytkowski MT, Ladenson PW. Age-related changes in basal and sodiumstimulated atrial and plasma atrial natriuretic factor (ANF) in the rat. J Gerontol 1991;46(3):B107-B110. https://doi.org/10.1093/geronj/46.3.B107 [27] Wu SQ, Kwan CY, Tang F. The effect of aging on ANP levels in the plasma, heart, and brain of rats. J Gerontol A Biol Sci Med Sci 1997;52(5):B250-B254. https://doi.org/10.1093/gerona/52A.5.B250 [28] Ohba H, Takada H, Musha H, et al. Effects of prolonged strenuous exercise on plasma levels of atrial natriuretic peptide and brain natriuretic peptide in healthy
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Figure Legends Fig. 1. Electronic photomicrograph showing nuclear pore (black arrow), Golgi apparatus (G); granules of ANP (white arrows) and BNP (arrow heads), mitochondria (M), endoplasmic reticulum (ER). Euchromatin (E) and nucleus (N) are also shown. Scale bar = 0.2 µm. Fig 2. (A) Electron micrographs of right atrial cardiomyocytes of both control and diabetic rats, in which natriuretic granules are observed in the cytoplasm (arrow) near the nucleus (N) and the mitochondria (M). Scale bar = 0.2 µm. Numerical density of both (B) ANP and (C) BNP granules. Diameter of both (D)
ANP and (E) BNP granules. Percentage of small (S), medium (M) and large (L) granules of (F) ANP and (G) BNP. Data are expressed as mean ± SEM. Fig. 3. Volume density of (A) mitochondria, (B) endoplasmic reticulum, and (C) Golgi complex in the CG and DG groups. Data are expressed as mean ± SEM.
Table 1. Systolic blood pressure (mmHg) and blood glucose (mg/dl) between the groups. Parameter Arterial pressure (mmHg) Glycemia (mg/dl)
CG
DG
P Value
113±2.9
112±2.1
0.1901
96±12
364±28*
0.0001
Data are expressed as mean ± SEM.
Highlights 1. The numerical density of ANP and BNP in cardiomyocytes was significantly higher in the diabetic elderly animals. 2. The ANP and BNP granule size was significantly lower in the diabetic group. 3. At the end of the experiment, diabetic animals remained normotensive. 4. The density of cytoplasmic organelles of the cardiomyocytes was not influenced by diabetes.
S1054-8807(19)30346-1
DOI:
https://doi.org/10.1016/j.carpath.2019.107181
Reference:
CVP 107181
To appear in:
Cardiovascular Pathology
Received Date: 23 September 2019 Revised Date:
10 October 2019
Accepted Date: 11 November 2019
Please cite this article as: Souza de Andrade N, Barros Chucata KL, Magalhães WV, Baptista Nucci RA, Da Costa-Santos N, Dias IR, Douglas de Souza Lima H, Mesiano Maifrino LB, Rodrigues de Souza R, Ultrastructural effects of diabetes in the right atrium cardiomyocytes of elderly Wistar rats, Cardiovascular Pathology, https://doi.org/10.1016/j.carpath.2019.107181. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Inc. All rights reserved.
Ultrastructural effects of diabetes in the right atrium cardiomyocytes of elderly Wistar rats Natalie Souza de Andrade1,2, Kemily Loren Barros Chucata1, Walkyria Villegas Magalhães1, Ricardo Aparecido Baptista Nucci1,3,*, Nicolas Da Costa-Santos1, Igor Roberto Dias1, Hunter Douglas de Souza Lima1, Laura Beatriz Mesiano Maifrino1,4, Romeu Rodrigues de Souza1,5 1
Department
of
Physical
Education,
Laboratory
of
Morphological
and
Immunohistochemical Studies, São Judas Tadeu University, São Paulo, SP, Brazil 2
Uninove University, São Paulo, SP, Brazil
3
Department of Pathology, University of São Paulo Medical School, São Paulo,
SP, Brazil 4
Dante Pazzanese Institute of Cardiology, São Paulo, SP, Brazil
5
Department of Anatomy, Institute of Biomedical Sciences of University of São
Paulo, São Paulo, SP, Brazil
Corresponding author at: São Judas Tadeu University and University of São Paulo Medical School, São Paulo, Brazil. E-mail address: [email protected] (R.A.B. Nucci)
ABSTRACT The present study aimed to evaluate the effects of diabetes on quantitative parameters of right atrial cardiomyocytes of elderly rats. Wistar rats (14-mo of age) were divided into two groups: streptozotocin-diabetic rats (DG); and control rats (CG). The groups were sacrificed at 16 months. Ultrafine sections of the right atrium were analyzed by electron microscopy. In elderly diabetic animals, histograms of the frequency distribution of natriuretic peptides according to their size showed increased number of small and medium peptides in relation to large peptides, which increased its numerical density leading to a decrease in the mean diameter of both natriuretic peptides. However, elderly diabetic animals remained normotensive. No significant difference was observed between the groups for the volume density of mitochondria, endoplasmic reticulum and Golgi apparatus. In conclusion, elderly diabetic rats showed increased functional activity of atrial cardiomyocytes with greater production of natriuretic peptides in association with a quantitative maintenance of cytoplasmic components. Key words: Aging; Atrial cardiomyocytes; Electron microscopy; Morphometry; Streptozotocin.
1. Introduction Diabetes mellitus (DM) have direct effects on cardiac muscle, and its effects on atrial cardiomyocytes have been studied in experimental models, e.g., rats [1-5]. Atrial cardiomyocytes produce atrial natriuretic peptide (ANP) and B-type or brain natriuretic peptide (BNP) that act to control blood pressure [6-8]. Although, the effects of DM on the production of ANP and BNP in atrial cardiomyocytes have been studied in young animals demonstrating an important relationship between cardiomyocyte function and these peptides [914], there is still a lack of studies with elderly diabetic rats [15]. Additionally, it is suggested that the production of both ANP and BNP in cardiomyocytes is directly related to the function of mitochondria, Golgi complex and endoplasmic reticulum, which, in turn, has an influence on the volume densities of these organelles [9,10,16]. However, the effects of DM on these components in elderly rats have not been elucidate. The present study aimed to evaluate the effects of diabetes on quantitative parameters of right atrial cardiomyocytes of elderly rats through morphometric techniques on electronic photomicrographs [17,18]. 2. Methods All experimental procedures conformed to the guiding principles of the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996. The Institutional Animal Care and Use Committee of the university approved the experimental protocol (protocol number 100/08). The animals used in the present study were male Wistar rats (14-month-old) purchased from the Laboratory Animal Center of Institute of Biomedical Sciences of University of São Paulo, São Paulo, Brazil. Animals were divided in two groups (n = 8/per group): control group (CG); and rats submitted to diabetes induction (DG). The animals were housed in plastic cages with access to food and water ad libitum and maintained on a 12 hours light/dark cycle at room temperature (23–26°C). The food stuff consumption of each rat per day and night was measured regularly.
2.1. Induction of DM DM was induced in the animals at age of 14 months by an intravenous injection of Streptozotocin (70 mg / kg, Sigma) after one overnight fast (8-10 hours) [4]. The control group received at age of 14 months only vehicle (10 mm citrate buffer, pH 4.5) after an overnight fast. After 30 days of induction to DM, a blood sample of the animals from the DG was collected and sent to the laboratory, and DM-compatible glycemic levels were found. 2.2. Euthanasia and sample collection At the end of the experiment (16 months age), the systolic pressure was obtained indirectly through the tail cuff method [19], and the animals had their final glycemia evaluated by glycosometer to comparison. Animals from both groups were euthanized with sodium pentobarbital and their ventricles perfused with heparinized saline solution (1 mg / 300 ml solution). The right atria were removed and reduced to fragments of approximately 4 mm³. The fragments were placed in glutaraldehyde 2% in sodium cacodylate buffer solution (0.2 m, pH 7.3) for three hours. The fragments were then washed three times with the same buffer solution for 5 minutes at a time, and subsequently placed in a solution of 1% osmium tetroxide in sodium buffer and cacodylate for 2 hours. The fragments remained overnight in 0.5% uranyl acetate with sucrose (54 mg / 100 ml) and after washing with the buffer were dehydrated in the growing series and absolute alcohol and propylene oxide for 8 hours under rotation. The fragments were then included in pure resin for 5 hours and finally left in the same resin at 60°C for 5 days. The ultr afine sections were obtained with a diamond knife in an ultramicrotome (Sorvall MT-2), and after having been contrasted with uranyl acetate and lead citrate were used to perform cardiomyocyte photomicrographs in the transmission electron microscope (Jeol) of the Institute of Biomedical Sciences of University of São Paulo. 2.3. Morphometric evaluation Electron micrographs of ten areas per animal, i.e., a total of 160 areas, sampled by random [20], were taken with an increase of ×15000. We used a quadrangular area of 54 µm2 for counting ANP and BNP numerical density in the cardiomyocyte. Using a Zeiss microscope, coupled to a computerized image analysis program (Axio Vision, Zeiss) from the Laboratory of Morphological and
Immunohistochemical Studies of the São Judas Tadeu University, we measured both ANP and BNP granule diameters. The largest and smallest diameter of each granule were measured, and the arithmetic averages of both measurements were used for the final calculations. With the aid of ImageJ software, we analyzed the volume density (%) of mitochondria, endoplasmic reticulum and Golgi apparatus in the cytoplasm (Fig. 1). The volume density corresponds to the area occupied by the structure in the field in percentage [17]. It was obtained using a test system equipped with 80 points (considered as 100%) placed on each photomicrograph. The following formula was used to obtain the volume density of the cardiomyocyte components: Vv [structure] = ΣP [structure] 100 / TP; where, Vv [structure] = Volume density of the structure, ΣP [structure] = number of points on the structure in question, and TP = Total number of points (80) of the test system [17,18]. 2.5. Statistical analysis Data were expressed as mean ± standard error (SEM). We conducted unpaired Student’s t-test to examine whether the groups were different regarding the variables. The statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Prism, Inc., San Diego, CA). The alpha level was set at the 0.05 level, and all tests were two-tailed. 3. Results 3.1. Values of systolic blood pressure and blood glucose Table 1 shows the final measurement of systolic blood pressure and glycemia of the animals in both groups before euthanasia. We verified that there was no significant difference between the arterial pressure values between the groups (p>0.05). However, the glycemia values in the DG animals are compatible with the presence of diabetes (p<0.05). 3.2. Morphometry The number and diameter of the granules in the right atrium cardiomyocytes in both groups of rats are shown in Fig. 2. We observed that the values for the number of granules in DG are significantly higher than CG for both types of granules (ANP and BNP) (p<0.001). On the other hand, the
diameter of the granules of ANP and BNP were significantly lower in DG when compared to CG (p<0.001). The results obtained for the distribution of frequencies (%) of the granules of ANP and BNP according to their diameter in the groups CG and DG can be observed (Fig. 2). The results for ANP show that diabetes promoted a decrease in the percentage of medium and large granules, and an increase in the percentage of small ones. In addition, the results for the BNP shows that there was a decrease in the percentage of small granules and an increase in the percentages of the medium and large granules in diabetic animals. However, Fig. 3 shows a not significant decrease (p>0.05) in the volume density of mitochondria, endoplasmic reticulum and Golgi complex in the diabetic animals when compared to control. Qualitatively, we observed preserved morphological aspects for these organelles. 4. Discussion There are two main findings in this work. First, the numerical density of ANP and BNP in cardiomyocytes was significantly higher in the diabetic elderly group compared to the control group, and the ANP and BNP granule size was significantly lower in the diabetic group than in the control group, indicating that diabetes had an influence in the production of natriuretic peptides. These results are similar to previous studies on experimental induction of DM in young animals and in diabetic patients [21-23]. Second, despite previous studies had demonstrate lesions on cytoplasmic organelles [24,25], we observed in elderly streptozotocin-diabetic rats a nonsignificant influence of this disease on the density of cardiomyocytes organelles. Additionally, these organelles were wellpreserved in both groups. According to previous works, during aging there is an increase of natriuretic peptides [26,27]. In our study, we observed that diabetes increased the numerical density of natriuretic peptides when compared to aged nondiabetic animals. The increased density of ANP and BNP granules observed in the cytoplasm of cardiomyocytes from diabetic animals, may be due to an increase in peptide production or a decrease in their secretion into the bloodstream. To corroborate with our results, a previous study in mice showed
that the secretion of these peptides was reduced in diabetic animals [21], suggesting that in diabetes, polyuria occurs with glycosuria due to hyperglycemia. It is possible that diabetic cardiomyocytes retain ANP and BNP in cardiomyocytes cytoplasm to avoid excessive loss of sodium, as these peptides, when released into the bloodstream, act in the kidneys increasing the output of sodium and water [6,16,28]. Thus, in animals with diabetes, the reduction of ANP and BNP secretion may occur to maintain extracellular sodium rates. Regarding the diameter, diabetic animals had smaller granules than those of the control animals. Furthermore, DM increased smaller ANP and medium BNP granules, which explains the decrease in mean diameter observed in the granules of diabetic animals. These results suggest that DM influenced not only the size but also the proportion of the granules in terms of their size and future studies are need to elucidate the mechanisms related to this phenomenon. We observed that diabetic animals did not show presence of arterial hypertension, which suggests that they remained normotensive. Thus, the increase of ANP and BNP production by cardiomyocytes was not due to the presence of hypertension, as both peptides have an important role in maintaining blood pressure [6], but exclusively to diabetes. Mitochondrial lesions were observed in diabetic rats indicating failure of mitochondrial control mechanisms [25]. Studies have related mitochondrial function to the balance between energy demand and nutrient supply, suggesting that changes in mitochondria may act as a mechanism for bioenergetic adaptation during pathological cardiac remodeling [25,29]. Additionally, Kordowiak et al. [24] observed a reduction of Golgi complex membranes and endoplasmic reticulum in hepatic cells of diabetic rats. In contrast, our study showed that the mitochondria, Golgi apparatus and endoplasmic reticulum of cardiomyocytes did not undergo quantitative changes in the prevalence of DM, however, our results may be related to the period of diabetes (2 months) that the animals underwent. However, our results should be examined considering the study limitations. Although, cardiomyocytes organelles were well-preserved, we did not analyzed the activity of these organelles [25]. On the other hand, future
studies should consider the period of diabetes for a better understanding of our results, as previous studies with streptozotocin-diabetic experimental models had a period above 2 months [1,4]. Although our study has limitations, we also have some advantages, as we presented ultrastructural effects of diabetes in the right atrium using standardized morphometrical techniques [17,18]. Additionally, our diabetic experimental model was in accordance with previous described in the literature [1-4]. In conclusion, elderly diabetic rats showed increased functional activity of atrial cardiomyocytes with greater production of ANP and BNP granules in association with a quantitative maintenance of cytoplasmic components. Accordingly, further studies are needed regarding the physiological and molecular mechanisms involving cardiomyocytes injury and diabetes for a better understanding of our results. Declaration of interest The authors declare not competing or financial interest. References [1] Hussein AEAM, Omar NM, Sakr H, Elsamanoudy AZ, Shaheen D. Modulation of metabolic and cardiac dysfunctions by insulin sensitizers and angiotensin receptor blocker in rat model of type 2 diabetes mellitus. Can J Physiol Pharmacol 2011;89(3):216-226. https://doi.org/10.1139/Y11-012 [2] Kato T, Yamashita T, Sekiguchi A, et al. Angiotensin II type 1 receptor blocker attenuates diabetes-induced atrial structural remodeling. J Cardiol 2011;58(2):131-136. https://doi.org/10.1016/j.jjcc.2011.06.003 [3] Musial DC, de Magalhães Galvão K, Miranda-Ferreira R, et al. Alteration of purinergic neurotransmission in isolated atria of streptozotocin-induced diabetic rats.
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Figure Legends Fig. 1. Electronic photomicrograph showing nuclear pore (black arrow), Golgi apparatus (G); granules of ANP (white arrows) and BNP (arrow heads), mitochondria (M), endoplasmic reticulum (ER). Euchromatin (E) and nucleus (N) are also shown. Scale bar = 0.2 µm. Fig 2. (A) Electron micrographs of right atrial cardiomyocytes of both control and diabetic rats, in which natriuretic granules are observed in the cytoplasm (arrow) near the nucleus (N) and the mitochondria (M). Scale bar = 0.2 µm. Numerical density of both (B) ANP and (C) BNP granules. Diameter of both (D)
ANP and (E) BNP granules. Percentage of small (S), medium (M) and large (L) granules of (F) ANP and (G) BNP. Data are expressed as mean ± SEM. Fig. 3. Volume density of (A) mitochondria, (B) endoplasmic reticulum, and (C) Golgi complex in the CG and DG groups. Data are expressed as mean ± SEM.
Table 1. Systolic blood pressure (mmHg) and blood glucose (mg/dl) between the groups. Parameter Arterial pressure (mmHg) Glycemia (mg/dl)
CG
DG
P Value
113±2.9
112±2.1
0.1901
96±12
364±28*
0.0001
Data are expressed as mean ± SEM.
Highlights 1. The numerical density of ANP and BNP in cardiomyocytes was significantly higher in the diabetic elderly animals. 2. The ANP and BNP granule size was significantly lower in the diabetic group. 3. At the end of the experiment, diabetic animals remained normotensive. 4. The density of cytoplasmic organelles of the cardiomyocytes was not influenced by diabetes.